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Increasing evidence demonstrated the oral microbial community profile characteristics affected by conventional cigarettes smoking, but few studies focus on oral microbiome in response to electronic cigarettes (E-cigarettes). This study aimed to investigate the effect of E-cigarettes on the oral microbiome and to describe the difference of oral community profiles between E-cigarette smokers and tobacco smokers. 16S rRNA V4 gene sequencing was performed to investigate the oral microbial profiles of 5 E-cigarette smokers, 14 tobacco smokers, 8 quitting tobacco smokers, and 6 nonsmokers. The Chao1, ACE, and Shannon diversity indexes increased significantly in saliva samples collected from E-cigarette smokers and tobacco smokers compared to the non-smokers, and no significant difference was found in alpha diversity between E-cigarette smokers and tobacco smokers. The main phyla Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria and major genera Neisseria, Streptococcus, Prevotellaceae, Fusobacterium, and Porphyromonas dominated in the smoking groups, while Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria became the dominant phyla along with the genera Corynebacterium, Neisseria, Streptococcus, Actinomyces, and Porphyromonas in the nonsmokers. The differences in the phylum Actinobacteria and genus Corynebacterium contributed to various functional differences between smokers and nonsmokers. The difference on oral microbial and composition between E-cigarettes and common tobacco were associated with increased Prevotellaceae and decreased Neisseria. Additionally, smoking cessation could lead to re-establishment of the oral microbiome to that of nonsmokers. Our data demonstrate that E-cigarette smoking had different effects on the structure and composition of the oral microbial community compared to tobacco smoking. However, the short- and long-term impact of E-cigarette smoking on microbiome composition and function needs further exploration.

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot- or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further functional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+-induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.

CRISPR/Cas9 genome-editing technology has revolutionized plant science and holds enormous promise for crop improvement. The exploration of this system received much attention regarding plant genome editing. Here, by editing the NtPDS gene in tobacco, we first verified that incorporating an OsU3-tRNA promoter combination into the CRISPR/Cas9 system contributed to the highest editing efficiency, as the sgRNA expression level was greater than that resulting from the AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter combination instead of AtU6 and by fusing an AtUb10-Ros1 expression cassette to the T-DNA to monitor the transgene events. The new system was named pOREU3TR. As expected, 49 transgene-free and homozygous gene-edited green plants were effectively screened in the T

2, 3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM), is a key enzyme that catalyzes the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis. iPGAM has been found to be involved in several physiological processes, including stomatal movement, chlorophyll synthesis, pollen production, chloroplast development and photosynthesis in different plants. However, the roles iPGAM plays in model plant tobacco are still elusive. In the current study, two copies of the iPGAM gene in Nicotiana tabacum were cloned, NtiPGAMa and NtiPGAMb, from the variety “Honghuadajinyuan”. Expression profiles and subcellular localization analyses showed that NtiPGAMa and NtiPGAMb were highly expressed in the anthers and were localized to the nucleus, cytosol and cell membrane. Using the CRISPR/Cas9 system, three mutant lines with mosaic leaves were obtained by targeting the second exon of the iPGAM gene. Based on Sanger sequencing, the mutant lines were chimeric plants and displayed mutation rates ranging from 82.3% to 92.5%. The iPGAM mutations showed significantly decreased photosynthetic pigments, i.e., chlorophyll a, chlorophyll b and carotenoids, thinning or loss of chloroplast thylakoid lamellar structures. RT-PCR analysis showed that the expressions of genes associated with chlorophyll synthesis and photosynthesis were significantly down-regulated in the iPGAM mutant plants compared to wild type. The pollen development was greatly impaired in mutant plants, resulting in reduced and deformed pollens with grains inviable. Our findings indicate that iPGAM is critical for pollen vitality, chlorophyll synthesis and chloroplast morphogenesis in N. tabacum.

Background: CRISPR-Cas9 Genome-editing technology has revolutionized the plant science and hold enormous promise in crop improvement. When performing the gene editing system for crops, it is crucial to eliminate of the Cas9/sgRNA T-DNA cassette in the T1 generation. Results: Here, we firstly validated that incorporating an OsU3-tRNA promoter combination in the CRISPR/Cas9 system contributed to the highest mutagenesis efficiency that increased sgRNA expression levels over the AtU6-tRNA and AtU6 promoters by editing the NtPDS gene (Ntab0595110) in tobacco. Then we optimized the existing tobacco CRISPR/Cas9 system by using the OsU3-tRNA promoter combination instead of AtU6, and fusing an AtUb10-Ros1 expression cassette in T-DNA for monitoring the transgene events. The new vector was named as pOREU3TR. As expected, 52 transgene-free and homozygous gene-edited green plants were effectively screened at T1 generation by editing the NtLHT1 gene (Ntab0818090) in tobacco, and the contents of most free amino acids in the T2 mutants ntlht1 leaves were detected significantly different from those in the wild type leaves, demonstrating the highly efficient of the system.Conclusions: This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements to increase the efficiency of plant genome editing.

Sugars are both nutrients and important signal molecules in higher plants. Sugar transporters (STs) are involved in sugar loading and unloading and facilitate sugar transport across membranes. Tobacco (Nicotiana tabacum) is a model plant and one of the most significant plants economically. In our research, 92 N. tabacum ST (NtST) genes were identified and classified into eight distinct subfamilies in the tobacco genome based on phylogenetic analysis. Exon-intron analysis revealed that each subfamily manifested closely associated gene architectural features based on a comparable number or length of exons. Tandem repetition and purifying selection were the main factors of NtST gene evolution. A search for cis-regulatory elements in the promoter sequences of the NtST gene families suggested that they are probably regulated by light, plant hormones, and abiotic stress factors. We performed a comprehensive expression study in different tissues, viarious abiotic and phytohormone stresses. The results revealed different expression patterns and the functional diversification of NtST genes. The resulting data showed that NtSFP1 was highly expressed all measured five tobacco tissues, and also regulated by the MeJA, and temperature stress. In addition, the virus-induced NibenSFP1 silencing in tobacco and detected dramatically enhanced glucose content, indicating the NtSFP1 might regulate the glucose content and involved in MeJA signaling way to response the temperature stress. In general, our findings provide useful information on understanding the roles of STs in phytohormone signaling way and abiotic stresses in N. tabacum.

Previous studies have found that prenatal morphine (PNM) exposure leads to both increased and decreased risk of substance abuse in offspring. Understanding more about the neurobiological changes after the PNM exposure would help to understand more about this issue. Signaling from dopamine neurons of the ventral tegmental area (VTA) in the mesoaccumbal and mesocortical pathways plays a vital role in drug dependency. To provide further knowledge about the effects of PNM on drug seeking behavior and the dopamine system. We recorded local field potentials (LFP) simultaneously in the VTA, NAc (nucleus accumbens), BLA (basolateral amygdala) and mPFC (medial prefrontal cortex) in male adult rats prenatally treated with saline or morphine. Morphine (10 mg/kg) induced conditioned place preference (CPP) establishment, extinction and priming were tested to investigate the effects of PNM on addictive-like behavior. In addition, the expression of nuclear histone deacetylases (HDAC4, HDAC5), which plays essential epigenetic roles in neuroplasticity after drug use were also tested in VTA and NAc. The results showed that PNM did not change the acquisition of morphine CPP in male rats, but impaired CPP extinction and morphine (5 mg/kg) - primed reinstatement of CPP after extinction. PNM increased the low gamma (30-60 Hz) and high (60-90 Hz) gamma LFP powers in NAc and BLA. PNM also leads to increased theta (4-9 Hz) coherence between VTA and NAc, and increased HDAC5 expression in VTA. After chronic morphine administration, coherence between VTA-NAc, mPFC-NAc and mPFC-BLA increased significantly in PNS rats, but no changes were find in PNM rats, indicating impaired plasticity in brain circuits. All these results suggest that PNM exposure increased reward processing in adult male rats, but impaired morphine CPP extinction and reinstatement, which relate to decreases network plasticity and increased HDAC5 expression in the reward system.

Major depressive disorder (MDD) is the most prevalent mental disorder that affects more than 200 million people worldwide. Recent large-scale genome-wide association studies (GWAS) have identified multiple risk variants that show robust association with MDD. Nevertheless, how the identified risk variants confer risk of MDD remains largely unknown. To identify risk variants that are associated with gene expression in human brain and to identify genes whose expression change may contribute to the susceptibility of MDD, we systematically integrated the genetic associations from a large-scale MDD GWAS (N = 480,359) and brain expression quantitative trait loci (eQTL) data (N = 494) using a Bayesian statistical framework (Sherlock). Sherlock integrative analysis showed that FLOT1 was significantly associated with MDD (P = 6.02 × 10(−6)), suggesting that risk variants may contribute to MDD susceptibility through affecting FLOT1 expression. We further examined the expression level of FLOT1 in MDD cases and controls and found that FLOT1 was significantly upregulated in brains and peripheral blood of MDD cases compared with controls (European sample). Interestingly, we found that FLOT1 expression was also significantly upregulated in peripheral blood of first-episode drug-naive MDD cases compared with controls (P = 1.01 × 10(−7), Chinese sample). Our study identified FLOT1 as a novel MDD risk gene whose expression level may play a role in MDD. In addition, our findings also suggest that risk variants may confer risk of MDD through affecting expression of FLOT1. Further functional investigation of FLOT1 may provide new insights for MDD pathogenesis.

Stable intracellular and intercellular osmolarity is vital for all physiological processes. Although it is the first organ that receives food, the osmolarity around the mouth epithelium has never been systematically investigated. We found that oral epithelial cells are a population of ignored cells routinely exposed to hypertonic environments mainly composed of saline, glucose, etc. in vivo after chewing food. By using cultured oral epithelial cells as an in vitro model, we found that the hypotonic environments caused by both high NaCl and high glucose induced cell death in a dose- and time-dependent manner. Transcriptomics revealed similar expression profiles after high NaCl and high glucose stimulation. Most of the common differentially expressed genes were enriched in “mitophagy” and “autophagy” according to KEGG pathway enrichment analysis. Hypertonic stimulation for 1 to 6 h resulted in autophagosome formation. The activation of autophagy protected cells from high osmolarity-induced cell death. The activation of Hsp70 by the pharmacological activator handelin significantly improved the cell survival rate after hypertonic stimulation. The protective role of Hsp70 activation was partially dependent on autophagy activation, indicating a crosstalk between Hsp70 and autophagy in hypertonic stress response. The extract of the handelin-containing herb Chrysanthemum indicum significantly protected oral epithelial cells from hypertonic-induced death, providing an inexpensive way to protect against hypertonic-induced oral epithelial damage. In conclusion, the present study emphasized the importance of changes in osmolarity in oral health for the first time. The identification of novel compounds or herbal plant extracts that can activate autophagy or HSPs may contribute to oral health and the food industry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12192-020-01068-2) contains supplementary material, which is available to authorized users.

Elongator protein 3 (Elp3) is the enzymatic unit of the elongator protein complex, a histone acetyltransferase complex involved in transcriptional elongation. It has long been shown to play an important role in cell migration; however, the underlying mechanism is unknown. Here, we showed that Elp3 is expressed in pre-migratory and migrating neural crest cells in Xenopus embryos and knockdown of Elp3 inhibited neural crest cell migration. Interestingly, Elp3 binds Snail1 through its zinc-finger domain and inhibits its ubiquitination by β-Trcp without interfering with the Snail1/Trcp interaction. We showed evidence that Elp3-mediated stabilization of Snail1 was likely involved in the activation of N-cadherin in neural crest cells to regulate their migratory ability. Our findings provide a new mechanism for the function of Elp3 in cell migration through stabilizing Snail1, a master regulator of cell motility.

The evolutionarily conserved Dbx homeodomain-containing proteins play important roles in the development of vertebrate central nervous system. In mouse, Dbx and Nkx6 have been suggested to be cross-repressive partners involved in the patterning of ventral neural tube. Here, we have isolated Xenopus Dbx2 and studied its developmental expression and function during neural development. Like XDbx1, from mid-neurula stage on, XDbx2 is expressed in stripes between the primary motoneurons and interneurons. At the tailbud stages, it is detected in the middle region of the neural tube. XDbx2 acts as a transcriptional repressor in vitro and over-expression of XDbx2 inhibits primary neurogenesis in Xenopus embryos. Over-expression of XDbx genes represses the expression of XNkx6.2 and vise versa. Knockdown of either XDbx1, XDbx2 or both by specific morpholinos induces lateral expansion of XNkx6.2 expression domains. These data reveal conserved roles for Dbx in primary neurogenesis and dorsoventral neural patterning in Xenopus.

Modification of proteins by ubiquitination plays important roles in various cellular processes. During this process, the target specificity is determined by ubiquitin ligases. Here we identify RNF220 (RING finger protein 220) as a novel ubiquitin ligase for Sin3B. As a conserved RING protein, RNF220 can bind E2 and mediate auto-ubiquitination of itself. Through a yeast two-hybrid screen, we isolated Sin3B as one of its targets, which is a scaffold protein of the Sin3/HDAC (histone deacetylase) corepressor complex. RNF220 specifically interacts with Sin3B both in vitro and in vivo. Sin3B can be regulated by the ubiquitin-proteasome system. Co-expression of RNF220 promotes the ubiquitination and proteasomal degradation of Sin3B. Taken together, these results reveal a new mechanism for regulating the Sin3/HDAC complex.

To study the infection status of Helicobacter pylori (H. pylori) and sensitivity to commonly used antibiotics in Taizhou district,Zhejiang province.39 099 cases aged between 5 and 95 years old (mean as 48.42 years) were involved during January 2010 to December, 2013 for this study. Sex ratio was 1 : 0.95. Yearly distribution of the number of cases were 5 031, 6 709, 11 902 and 15 457 in 2010, 2011, 2012 and 2013, respectively. Gastric mucosal specimens were collected and H. pylori strains were isolated and cultured in the same platform in Zhiyuan Medical Inspection Institute of Hangzhou. Resistance tests of all the H. pylori isolates were performed to 6 commonly used antibiotics:metronidazole, clarithromycin, amoxicillin, gentamicin, levofloxacin and furazolidone with the agar dilution method. The antibiotic resistance rates of H. pylori strains isolated during year 2010-2013 and the changing trends were analyzed.Resistance rates to levofloxacin and clarithromycin kept at higher level and the highest was in 2011 and then decreased in both 2012 and 2013 (P0.01). The resistance rates to both levofloxacin and clarithromycin reached the highest in 2011 (P0.01), and decreased thereafter, with no significant change in 2013 to 2012 (P0.05).Antibiotic resistance rate against metronidazole for HP isolate was highest. Resistance rate against amoxicillin and furazolidone, gentamicin was low. Clinical treatment should choose amoxicillin and furazolidone, gentamicin. The resistance rates to levofloxacin and clarithromycin had been seen at a significantly downward trend since 2011. However, the combined resistance rates to levofloxacin and clarithromycin did not seem to reduce since 2012.

Regulation of neuronal gene expression is critical to nervous system development. REST (RE1-silencing transcription factor) regulates neuronal gene expression through interacting with a group of corepressor proteins including REST corepressors (RCOR). Here we show that Xenopus RCOR2 is predominantly expressed in the developing nervous system. Through a yeast two-hybrid screen, we isolated Xenopus ZMYND8 (Zinc finger and MYND domain containing 8) as an XRCOR2 interacting factor. XRCOR2 and XZMYND8 bind each other in co-immunoprecipitation assays and both of them can function as transcriptional repressors. XZMYND8 is co-expressed with XRCOR2 in the nervous system and overexpression of XZMYND8 inhibits neural differentiation in Xenopus embryos. These data reveal a RCOR2/ZMYND8 complex which might be involved in the regulation of neural differentiation.