USP39 Promotes the Viability and Migration of Head and Neck Squamous Cell Carcinoma Cell by Regulating STAT1.

Objective: Ubiquitin-specific peptidase 39 (USP39) plays a carcinogenic role in many cancers, but little research has been conducted examining whether it is involved in head and neck squamous cell carcinoma (HNSCC). Therefore, this study explored the functional role of USP39 in HNSCC. Method: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins (DEPs) between the HNSCC tumor and adjacent healthy tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to assess the functional enrichment of DEPs. Immunohistochemistry was used to detect protein expression. The viability and migration of two HNSCC cell lines, namely CAL27 and SCC25, were detected using the cell counting kit-8 assay and a wound healing assay, respectively. Quantitative real-time PCR was used to detect the expression level of signal transducer and activator of transcription 1 (STAT1) mRNA. Results: LC-MS/MS results identified 590 DEPs between HNSCC and adjacent tissues collected from 4 patients. Through GO and KEGG pathway analyses, 34 different proteins were found to be enriched in the spliceosome pathway. The expression levels of USP39 and STAT1 were significantly higher in HNSCC tumor tissue than in adjacent healthy tissue as assessed by LC-MS/MS analysis, and the increased expression of USP39 and STAT1 protein was confirmed by immunohistochemistry in clinical samples collected from 7 additional patients with HNSCC. Knockdown of USP39 or STAT1 inhibited the viability and migration of CAL27 and SCC25 cells. In addition, USP39 knockdown inhibited the expression of STAT1 mRNA in these cells. Conclusion: Our findings indicated that USP39 knockdown may inhibit HNSCC viability and migration by suppressing STAT1 expression. The results of this study suggest that USP39 may be a potential new target for HNSCC clinical therapy or a new biomarker for HNSCC.

A 5.2-kb insertion in the coding sequence of PavSCPL, a serine carboxypeptidase-like enhances fruit firmness in Prunus avium.

Fruit firmness is an important trait in sweet cherry breeding because it directly positively influences fruit transportability, storage and shelf life. However, the underlying genes responsible and the molecular mechanisms that control fruit firmness remain unknown. In this study, we identified a candidate gene, PavSCPL, encoding a serine carboxypeptidase-like protein with natural allelic variation, that controls fruit firmness in sweet cherry using map-based cloning and functionally characterized PavSCPL during sweet cherry fruit softening. Genetic analysis revealed that fruit firmness in the 'Rainier' × 'Summit' F1 population was controlled by a single dominant gene. Bulked segregant analysis combined with fine mapping narrowed the candidate gene to a 473-kb region (7418778-7 891 914 bp) on chromosome 6 which included 72 genes. The candidate gene PavSCPL, and a null allele harbouring a 5244-bp insertion in the second exon that completely inactivated PavSCPL expression and resulted in the extra-hard-flesh phenotype, were identified by RNA-sequencing analysis and gene cloning. Quantitative RT-PCR analysis revealed that the PavSCPL expression level was increased with fruit softening. Virus-induced gene silencing of PavSCPL enhanced fruit firmness and suppressed the activities of certain pectin-degrading enzymes in the fruit. In addition, we developed functional molecular markers for PavSCPL and the Pavscpl5.2-k allele that co-segregated with the fruit firmness trait. Overall, this research identified a crucial functional gene for fruit firmness. The results provide insights into the genetic control and molecular mechanism of the fruit firmness trait and present useful molecular markers for molecular-assisted breeding for fruit firmness in sweet cherry.

Sweet cherry AP2/ERF transcription factor, PavRAV2, negatively modulates fruit size by directly repressing PavKLUH expression.

For sweet cherry, fruit size is one of the main targets in breeding programs owing to the high market value of larger fruits. KLUH/CYP78A5 is an important regulator of seed/fruit size in several plant species, but its molecular mechanism is largely unknown. In this study, we characterized the function of PavKLUH in the regulation of sweet cherry fruit size. The ectopic overexpression of PavKLUH in Arabidopsis increased the size of its siliques and seeds, whereas virus-induced gene silencing of PavKLUH in sweet cherry significantly decreased fruit size by restricting mesocarp cell expansion. We screened out an AP2/ERF transcription factor containing a B3-like domain, designated as PavRAV2, which was able to physically interact with PavKLUH promoter in a yeast one-hybrid (Y1H) system. In Y1H assays, electrophoretic mobility shift assays, and dual-luciferase reporter analyses, PavRAV2 directly bound to the promoter of PavKLUH in vitro and in vivo, and suppressed PavKLUH expression. Silencing of PavRAV2 resulted in enlarged fruit as a result of enhanced mesocarp cell expansion. Together, our results provide new insights into signaling pathways related to fruit size, and outline a possible mechanism for how the RAV transcription factor directly regulates CYP78A family members to influence fruit size and development.

Accurate Chromosome Identification in the Prunus Subgenus Cerasus (Prunus pseudocerasus) and its Relatives by Oligo-FISH.

A precise, rapid and straightforward approach to chromosome identification is fundamental for cytogenetics studies. However, the identification of individual chromosomes was not previously possible for Chinese cherry or other Prunus species due to the small size and similar morphology of their chromosomes. To address this issue, we designed a pool of oligonucleotides distributed across specific pseudochromosome regions of Chinese cherry. This oligonucleotide pool was amplified through multiplex PCR with specific internal primers to produce probes that could recognize specific chromosomes. External primers modified with red and green fluorescence tags could produce unique signal barcoding patterns to identify each chromosome concomitantly. The same oligonucleotide pool could also discriminate all chromosomes in other Prunus species. Additionally, the 5S/45S rDNA probes and the oligo pool were applied in two sequential rounds of fluorescence in situ hybridization (FISH) localized to chromosomes and showed different distribution patterns among Prunus species. At the same time, comparative karyotype analysis revealed high conservation among P. pseudocerasus, P. avium, and P. persica. Together, these findings establish this oligonucleotide pool as the most effective tool for chromosome identification and the analysis of genome organization and evolution in the genus Prunus.

The PavNAC56 transcription factor positively regulates fruit ripening and softening in sweet cherry (Prunus avium).

The rapid softening of sweet cherry fruits during ripening results in the deterioration of fruit quality. However, few genes related to sweet cherry fruit ripening and softening have been identified, and the molecular regulatory mechanisms underlying this process are poorly understood. Here, we identified and functionally characterized PavNAC56, a NAC transcription factor that positively regulates sweet cherry fruit ripening and softening. Gene expression analyses showed that PavNAC56 was specifically and abundantly expressed in the fruit, and its transcript levels increased in response to abscisic acid (ABA). A subcellular localization analysis revealed that PavNAC56 is a nucleus-localized protein. Virus-induced gene silencing of PavNAC56 inhibited fruit ripening, enhanced fruit firmness, decreased the contents of ABA, anthocyanins, and soluble solids, and down-regulated several fruit ripening-related genes. Yeast one-hybrid and dual-luciferase assays showed that PavNAC56 directly binds to the promoters of several genes related to cell wall metabolism (PavPG2, PavEXPA4, PavPL18, and PavCEL8) and activates their expression. Overall, our findings show that PavNAC56 plays an indispensable role in controlling the ripening and softening of sweet cherry fruit and provides new insights into the regulatory mechanisms by which NAC transcription factors affect nonclimacteric fruit ripening and softening.

Comparative Analysis of Transposable Elements and the Identification of Candidate Centromeric Elements in the Prunus Subgenus Cerasus and Its Relatives.

The subgenus Cerasus and its relatives include many crucial economic drupe fruits and ornamental plants. Repetitive elements make up a large part of complex genomes, and some of them play an important role in gene regulation that can affect phenotypic variation. However, the variation in their genomes remains poorly understood. This work conducted a comprehensive repetitive sequence identification across the draft genomes of eight taxa of the genus Prunus, including four of the Prunus subgenus Cerasus (Prunus pseudocerasus, P. avium, P. yedoensis and P. × yedoensis) as well as congeneric species (Prunus salicina, P. armeniaca, P. dulcis and P. persica). Annotation results showed high proportions of transposable elements in their genomes, ranging from 52.28% (P. armeniaca) to 61.86% (P. pseudocerasus). The most notable differences in the contents of long terminal repeat retrotransposons (LTR-RTs) and tandem repeats (TRs) were confirmed with de novo identification based on the structure of each genome, which significantly contributed to their genome size variation, especially in P. avium and P.salicina. Sequence comparisons showed many similar LTR-RTs closely related to their phylogenetic relationships, and a highly similar monomer unit of the TR sequence was conserved among species. Additionally, the predicted centromere-associated sequence was located in centromeric regions with FISH in the 12 taxa of Prunus. It presented significantly different signal intensities, even within the diverse interindividual phenotypes for Prunus tomentosa. This study provides insight into the LTR-RT and TR variation within Prunus and increases our knowledge about its role in genome evolution.

A Sweet Cherry Glutathione S-Transferase Gene, PavGST1, Plays a Central Role in Fruit Skin Coloration.

Sweet cherry, an economically important horticultural crop, has strong antioxidant activity. The fruits contain compounds potentially beneficial to human health-particularly anthocyanins, which are synthesized in cytosol and predominantly accumulated in vacuoles. Although anthocyanin levels differ among dark-red, blush, and yellow sweet cherry cultivars, the regulatory mechanism of anthocyanin transport and accumulation is not well understood in this species. In this study, we identified 53 glutathione S-transferase genes (PavGSTs) from sweet cherry and found that PavGST1 expression was well correlated with anthocyanin accumulation in cultivars with different fruit skin colors. TRV-mediated virus-induced silencing of PavGST1 decreased anthocyanin accumulation in sweet cherry fruits and downregulated the expressions of anthocyanin biosynthetic and regulatory genes. In addition, transient overexpression of PavGST1 promoted anthocyanin accumulation. Furthermore, yeast one-hybrid and dual-luciferase assays revealed that PavMYB10.1 and PavMYB75 directly bind to different MYB binding sites of the PavGST1 promoter (MBS-1 and MBS-3) to activate PavGST1 transcription. According to our results, PavGST1 plays a central role in sweet cherry fruit anthocyanin accumulation. Our findings provide novel insights into the coordinative regulatory mechanisms of PavGST1 and PavMYBs in anthocyanin accumulation in sweet cherry.

Evolution of Rosaceae Plastomes Highlights Unique Cerasus Diversification and Independent Origins of Fruiting Cherry.

Rosaceae comprises numerous types of economically important fruits, ornamentals, and timber. The lack of plastome characteristics has blocked our understanding of the evolution of plastome and plastid genes of Rosaceae crops. Using comparative genomics and phylogenomics, we analyzed 121 Rosaceae plastomes of 54 taxa from 13 genera, predominantly including Cerasus (true cherry) and its relatives. To our knowledge, we generated the first comprehensive map of genomic variation across Rosaceae plastomes. Contraction/expansion of inverted repeat regions and sequence losses of the two single-copy regions underlie large genomic variations in size among Rosaceae plastomes. Plastid protein-coding genes were characterized with a high proportion (over 50%) of synonymous variants and insertion-deletions with multiple triplets. Five photosynthesis-related genes were specially selected in perennial woody trees. Comparative genomic analyses implied divergent evolutionary patterns between pomaceous and drupaceous trees. Across all examined plastomes, unique and divergent evolution was detected in Cerasus plastomes. Phylogenomic analyses and molecular dating highlighted the relatively distant phylogenetic relationship between Cerasus and relatives (Microcerasus, Amygdalus, Prunus, and Armeniaca), which strongly supported treating the monophyletic true cherry group as a separate genus excluding dwarf cherry. High genetic differentiation and distinct phylogenetic relationships implied independent origins and domestication between fruiting cherries, particularly between Prunus pseudocerasus (Cerasus pseudocerasus) and P. avium (C. avium). Well-resolved maternal phylogeny suggested that cultivated P. pseudocerasus originated from Longmenshan Fault zone, the eastern edge of Himalaya-Hengduan Mountains, where it was subjected to frequent genomic introgression between its presumed wild ancestors and relatives.

Iodine monitoring models contribute to avoid adverse birth outcomes related more than adequate iodine intake.

BACKGROUND: Iodine plays an important role in pregnancy. How to maintain adequate iodine intake amongst pregnant women in each trimester of pregnancy to prevent adverse birth outcomes in central China is a challenge for clinical practice. METHODS: 870 pregnant women and their infants were enrolled in the study. Urinary iodine concentration (UIC) was measured using an inductively coupled plasma mass spectrometry (ICP-MS). Maternal and newborn information were obtained during follow-up. Multinomial logistic regression models were established. RESULTS: Median UIC of pregnant women was 172 ± 135 µg/L which is currently considered to be sufficient. Multivitamin supplements containing iodine, iodized salt intake and frequent milk intake were significantly associated with higher UIC. Multivariate logistic regression analysis showed that multivitamin supplements containing iodine and milk consumption were risk factors for more than adequate iodine (UIC ≥ 250 µg/L). Iodine-rich diet was significantly related to heavier birthweight, larger head circumference and longer femur length of the newborns while more than adequate iodine intake (UIC ≥ 250 µg/L) was a risk factor for macrosomia. Logistic regression models based on potential risk factors involving iodine containing supplements and iodine-rich diet were established to predict and screen pregnant women with high risk of more than adequate iodine intake among local pregnant women in different trimesters and guide them to supplement iodine reasonably to prevent the risk. CONCLUSIONS: Multivitamin supplements containing iodine and milk consumption were risk factors for maternal UIC ≥ 250 µg/L which was a risk factor for macrosomia. Iodine monitoring models were established to provide guidance for pregnant women to reduce the risk of more than adequate iodine intake, thereby contributing to reduce the risk of having a macrosomia.

PaMADS7, a MADS-box transcription factor, regulates sweet cherry fruit ripening and softening.

E-class MADS-box transcription factors, SEPALLATA (SEP) genes have an important role in floral organ initiation and development and fruit ripening. In this study, four sweet cherry SEP-like genes (PaMADS2, PaMADS4, PaMADS5, and PaMADS7) were cloned and functionally characterized. Gene expression analysis showed that the differential expression levels of PaMADS4 and PaMADS7 coincided with fruit ripening, and expression of PaMADS2 and PaMADS5 did not. Expression of PaMADS7 was affected by ABA and IAA. Subcellular localization assay demonstrated that four sweet cherry SEP-like proteins were all localized inside the nucleus. Silencing PaMADS7 using TRV-mediated virus-induced gene silencing inhibited fruit ripening and influenced major ripening-related physiological processes, such as ABA content, soluble sugar contents, fruit firmness, and anthocyanin content, as well as expression of ripening-related genes. In addition, silencing of PaMADS7 induced phenotype defects that suppressed fruit ripening, which were rescued by exogenous ABA. Furthermore, yeast one-hybrid assay (Y1H) and transient expression analyses revealed that PaMADS7 directly binds to the promoter of PaPG1, which is involved in sweet cherry fruit softening, and positively activated PaPG1expression. These results showed that PaMADS7 is an indispensable positive regulator of sweet cherry fruit ripening and softening.

Association between variants in vitamin D-binding protein gene and vitamin D deficiency among pregnant women in china.

BACKGROUND: The prevalence of vitamin D deficiency and insufficiency is extremely high in pregnant women worldwide. However, the association between single nucleotide polymorphisms (SNPs) in vitamin D metabolic pathway genes and 25-hydroxyvitamin D (25(OH)D) concentration among Chinese pregnant women is seldom reported. The risk of adverse neonatal outcomes due to maternal vitamin D deficiency has not been well investigated. METHODS: A total of 815 pregnant women and 407 infants were enrolled in this study. Serum 25(OH)D concentration was detected. DNA was extracted from the maternal blood for genotyping genetic SNPs in vitamin D pathway. An XGBoost model was established based on SNPs combined with external variables. RESULTS: Mean serum 25(OH)D level was 15.67 ± 7.98 ng/mL among the pregnant women. Seventy-five percent of pregnant women had 25(OH)D deficiency in China. SNPs of GC (rs17467825, rs4588, rs2282679, rs2298850, and rs1155563) were significantly associated with maternal 25(OH)D concentration. The influence of variants of rs17467825, rs4588, rs2282679, and rs2298850 on maternal 25(OH)D might be modified by vitamin D supplementation and sunshine exposure. An XGBoost model was established for monitoring 25(OH)D status in pregnant women and provided clinical advice to reduce the risk of 25(OH)D deficiency. Mothers with 25(OH)D deficiency hinted a risk for macrosomia. CONCLUSION: A high prevalence of vitamin D deficiency in China has been confirmed. A clinical model was established to guide pregnant women to supplement vitamin D according to genotype. Furthermore, we suggest the effect of maternal vitamin D status on the risk of macrosomia.

Acoustic Trauma Changes the Parvalbumin-Positive Neurons in Rat Auditory Cortex.

Acoustic trauma is being reported to damage the auditory periphery and central system, and the compromised cortical inhibition is involved in auditory disorders, such as hyperacusis and tinnitus. Parvalbumin-containing neurons (PV neurons), a subset of GABAergic neurons, greatly shape and synchronize neural network activities. However, the change of PV neurons following acoustic trauma remains to be elucidated. The present study investigated how auditory cortical PV neurons change following unilateral 1 hour noise exposure (left ear, one octave band noise centered at 16 kHz, 116 dB SPL). Noise exposure elevated the auditory brainstem response threshold of the exposed ear when examined 7 days later. More detectable PV neurons were observed in both sides of the auditory cortex of noise-exposed rats when compared to control. The detectable PV neurons of the left auditory cortex (ipsilateral to the exposed ear) to noise exposure outnumbered those of the right auditory cortex (contralateral to the exposed ear). Quantification of Western blotted bands revealed higher expression level of PV protein in the left cortex. These findings of more active PV neurons in noise-exposed rats suggested that a compensatory mechanism might be initiated to maintain a stable state of the brain.

PaCYP78A9, a Cytochrome P450, Regulates Fruit Size in Sweet Cherry (Prunus avium L.).

Sweet cherry (Prunus avium L.) is an important fruit crop in which fruit size is strongly associated with commercial value; few genes associated with fruit size have, however, been identified in sweet cherry. Members of the CYP78A subfamily, a group of important cytochrome P450s, have been found to be involved in controlling seed size and development in Arabidopsis thaliana, rice, soybean, and tomato. However, the influence of CYP78A members in controlling organ size and the underlying molecular mechanisms in sweet cherry and other fruit trees remains unclear. Here, we characterized a P. avium CYP78A gene PaCYP78A9 that is thought to be involved in the regulation of fruit size and organ development using overexpression and silencing approaches. PaCYP78A9 was significantly expressed in the flowers and fruit of sweet cherry. RNAi silencing of PaCYP78A9 produced small cherry fruits and PaCYP78A9 was found to affect fruit size by mediating mesocarp cell proliferation and expansion during fruit growth and development. Overexpression of PaCYP78A9 in Arabidopsis resulted in increased silique and seed size and PaCYP78A9 was found to be highly expressed in the inflorescences and siliques of transgenic plants. Genes related to cell cycling and proliferation were downregulated in fruit from sweet cherry TRV::PaCYP78A9-silencing lines, suggesting that PaCYP78A9 is likely to be an important upstream regulator of cell cycle processes. Together, our findings indicate that PaCYP78A9 plays an essential role in the regulation of cherry fruit size and provide insights into the molecular basis of the mechanisms regulating traits such as fruit size in P. avium.

The rice nuclear gene WLP1 encoding a chloroplast ribosome L13 protein is needed for chloroplast development in rice grown under low temperature conditions.

Plastidial ribosome proteins (PRPs) form the major component of the plastidial ribosome. Here we describe a rice mutant named wlp1 (white leaf and panicles 1) selected from a population of tissue culture regenerants. The early seedling leaves of the mutant were albino, as was the immature panicle at heading, and the phenotype was more strongly expressed in plants exposed to low temperature conditions. Changes in the leaf pigmentation of the mutant were due to altered chlorophyll content and chloroplast development. Positional cloning of WLP1, followed by complementation and knock-down experiments, showed that it encodes a 50S ribosome L13 protein. The WLP1 protein localized to the chloroplast. WLP1 was mainly transcribed in green tissues and particularly abundantly in the early seedling leaves. In addition, the expression level of WLP1 was induced by the low temperature. The transcription pattern of a number of genes involved in plastidial transcription/translation and in photosynthesis was altered in the wlp1 mutants. These results reveal that WLP1 is required for normal chloroplast development, especially under low temperature conditions. This is the first report on the function of PRPs in rice.