OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus.

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

Penaeid shrimp counteract high ammonia stress by generating and using functional peptides from hemocyanin, such as HMCs27.

Agricultural and anthropogenic activities release high ammonia levels into aquatic ecosystems, severely affecting aquatic organisms. Penaeid shrimp can survive high ammonia stress conditions, but the underlying molecular mechanisms are unknown. Here, total hemocyanin and oxyhemocyanin levels decreased in Penaeus vannamei plasma under high ammonia stress. When shrimp were subjected to high ammonia stress for 12 h, 24 hemocyanin (HMC) derived peptides were identified in shrimp plasma, among which one peptide, designated as HMCs27, was chosen for further analysis. Shrimp survival was significantly enhanced after treatment with the recombinant protein of HMCs27 (rHMCs27), followed by high ammonia stress. Transcriptome analysis of shrimp hepatopancreas after treatment with or without rHMCs27 followed by high ammonia stress revealed 973 significantly dysregulated genes, notable among which were genes involved in oxidation and metabolism, such as cytochrome C, catalase (CAT), isocitrate dehydrogenase, superoxide dismutase (SOD), trypsin, chymotrypsin, glutathione peroxidase, glutathione s-transferase (GST), and alanine aminotransferase (ALT). In addition, levels of key biochemical indicators, such as SOD, CAT, and total antioxidant capacity (T-AOC), were significantly enhanced, whereas hepatopancreas malondialdehyde levels and plasma pH, NH3, GST, and ALT levels were significantly decreased after rHMCs27 treatment followed by high ammonia stress. Moreover, high ammonia stress induced hepatopancreas tissue injury and apoptosis, but rHMCs27 treatment ameliorated these effects. Collectively, the current study revealed that in response to high ammonia stress, shrimp generate functional peptides, such as peptide HMCs27 from hemocyanin, which helps to attenuate the ammonia toxicity by enhancing the antioxidant system and the tricarboxylic acid cycle to decrease plasma NH3 levels and pH.

Kruppel homolog 1 modulates ROS production and antimicrobial peptides expression in shrimp hemocytes during infection by the Vibrio parahaemolyticus strain that causes AHPND.

Shrimp aquaculture has been seriously affected by acute hepatopancreatic necrosis disease (AHPND), caused by a strain of Vibrio parahaemolyticus that carries the Pir toxin plasmids (V. parahaemolyticus (AHPND)). In this study, the transcription factor, Kruppel homolog 1-like of Peneaus vannamei (PvKr-h1), was significantly induced in shrimp hemocytes after V. parahaemolyticus (AHPND) challenge, suggesting that PvKr-h1 is involved in shrimp immune response. Knockdown of PvKr-h1 followed by V. parahaemolyticus (AHPND) challenge increased bacterial abundance in shrimp hemolymph coupled with high shrimp mortality. Moreover, transcriptome and immunofluorescence analyses revealed that PvKr-h1 silencing followed by V. parahaemolyticus (AHPND) challenge dysregulated the expression of several antioxidant-related enzyme genes, such as Cu-Zu SOD, GPX, and GST, and antimicrobial peptide genes, i.e., CRUs and PENs, and reduced ROS activity and nuclear translocation of Relish. These data reveal that PvKr-h1 regulates shrimps' immune response to V. parahaemolyticus (AHPND) infection by suppressing antioxidant-related enzymes, enhancing ROS production and promoting nuclei import of PvRelish to stimulate antimicrobial peptide genes expression.

The mechanisms and factors that induce trained immunity in arthropods and mollusks.

Besides dividing the organism's immune system into adaptive and innate immunity, it has long been thought that only adaptive immunity can establish immune memory. However, many studies have shown that innate immunity can also build immunological memory through epigenetic reprogramming and modifications to resist pathogens' reinfection, known as trained immunity. This paper reviews the role of mitochondrial metabolism and epigenetic modifications and describes the molecular foundation in the trained immunity of arthropods and mollusks. Mitochondrial metabolism and epigenetic modifications complement each other and play a key role in trained immunity.

The histidine phosphatase LHPP of Penaeus vannamei is involved in shrimp hemocytes apoptosis.

LHPP (Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase) is a protein histidine phosphatase that modulates a hidden posttranslational modification called histidine phosphorylation. LHPP also acts as a tumor suppressor, which plays a pivotal role in various cellular processes. However, whether LHPP participates in the regulation of invertebrate's immunity is still unknown. Here we characterized a LHPP homolog in P. vannamei (designated PvLHPP), with a 807 bp length of open reading frame (ORF) encoding a putative protein of 268 amino acids. Sequence analysis revealed that PvLHPP contains a typical hydrolase 6 and hydrolase-like domain, which was conserved from invertebrate to vertebrate. PvLHPP was ubiquitously expressed in tissues and induced in hemocyte and hepatopancreas by Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, indicating that PvLHPP participated in the immune responses. Moreover, silencing of PvLHPP followed by V. parahaemolyticus inhibited hemocyte apoptosis. This study enriches our current insight on shrimp immunity, and provides novel perspective to understand immune-regulatory role of PvLHPP.

The MEF2 homolog of Penaeus vannamei is essential for maintaining the WSSV latent infection.

White spot syndrome virus (WSSV) is a lethal shrimp pathogen that has a latent infection cycle. The latent virus can easily turn into an acute infection when the culture environment changes, leading to widespread shrimp mortality. However, the mechanism of WSSV latent infection is poorly understood. Bioinformatic analysis revealed that the promoters of WSSV latency-related genes (i.e., wsv151, wsv366, wsv403, and wsv427) contained putative myocyte enhancer factor 2 (MEF2) binding sites. This suggested that the transcription factor MEF2 may be involved in WSSV latent infection. To further investigate this, a MEF2 homolog (PvMEF2) was cloned from Penaeus vannamei and its role in WSSV latent infection was explored. The results showed that knockdown of PvMEF2 led to an increase in the copy number of WSSV, indicating reactivation of WSSV from a latent infection. It was further demonstrated that suppression of PvMEF2 significantly decreased expression of the viral latency-related genes in WSSV-latent shrimp, while overexpression of PvMEF2 in Drosophila S2 cells activated the promoter activity of the viral latency-related gene. Additionally, we demonstrated that silencing of PvMEF2 was able to upregulate the expression of pro-apoptosis genes, thereby promoting cell apoptosis during latent infection. Collectively, the present data suggest that PvMEF2 could promote the expression of virus latency-related genes and enhance cell survival to maintain WSSV latent infection. This finding would contribute to a better understanding of the maintenance mechanism of WSSV latent infection.

The transcription factor CSL homolog in Penaeus vannamei positively regulates the transcription of the hemocyanin small subunit gene.

Hemocyanin, a copper-containing respiratory protein, is abundantly present in hemolymph of arthropods and mollusks and performs a variety of immunological functions. However, the regulatory mechanisms of hemocyanin gene transcription remain largely unclear. Our previous work showed that knockdown of the transcription factor CSL, a component of the Notch signaling pathway, downregulated the expression of Penaeus vannamei hemocyanin small subunit gene (PvHMCs), indicating the involvement of CSL in regulating the PvHMCs transcription. In this study, we identified a CSL binding motif ("GAATCCCAGA", +1675/+1684 bp) in the core promoter of PvHMCs (designated as HsP3). Dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA) demonstrated that the CSL homolog in P. vannamei (PvCSL) could directly bind and activate the HsP3 promoter. Moreover, in vivo silencing of PvCSL significantly attenuated the mRNA and protein expression of PvHMCs. Finally, in response to Vibrio parahaemolyticus, Streptococcus iniae and white spot syndrome virus (WSSV) challenge, the transcript of PvCSL and PvHMCs showed a positive correlation, suggesting that PvCSL could also modulate the expression of PvHMCs upon pathogen stimulation. Taken together, our present finding is the first to demonstrate that PvCSL is a crucial factor in transcriptional control of PvHMCs.

The biofilm characteristics and management of skin flap infection following cochlear implantation.

Objective: This study aims to assess the frequency, bacteriology, biofilm characteristics and management of skin flap infection (SFI) following cochlear implantation (CI). Methods: The study enrolled 1,251 patients receiving CI in the First Affiliated Hospital of Fujian Medical University between August 2001 and March 2021. Scanning electron microscopy (SEM) was utilised to characterise the aetiology of infection. A proposed classification system was applied to optimise treatments for post-operative skin flap infection. Results: After CI, SFI was reported in 16 patients (1.28%) and occurred more frequently in patients under 6 years of age. Of all SFI cases Staphylococcus aureus was the most common pathogen for flap infection, with 8 cases (50%) and bacterial biofilm was evident within the jelly-like substance on the surface of implanted devices in SFI patients. A two-stage classification was proposed to optimise the treatment schemes. Conservative therapy was recommended for stage I cases and surgical treatment for stage II patients. Conclusions: Paediatric patients are more susceptible to SFI after CI, which may be attributed to the formation of bacterial biofilm. The proposed classification can facilitate the management of SFI.

Shrimp Lipid Droplet Protein Perilipin Involves in the Pathogenesis of AHPND-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by a unique strain of Vibrio parahaemolyticus (Vp (AHPND)), has become the world's most severe debilitating disease in cultured shrimp. Thus far, the pathogenesis of AHPND remains largely unknow. Herein, in Litopenaeus vannamei, we found that a Vp (AHPND) infection significantly increased the expression of lipid droplets (LDs) protein LvPerilipin, as well as promoted the formation of LDs. In addition, the knockdown of LvPerilipin increased the shrimp survival rate in response to the Vp (AHPND) infection, and inhibited the proliferation of Vp (AHPND). Furthermore, we demonstrated that LvPerilipin depletion could increase the production of reactive oxygen species (ROS), which may be responsible for the decreased Vp (AHPND) proliferation. Taken together, our current data for the first time reveal that the shrimp lipid droplets protein Perilipin is involved in the pathogenesis of Vp (AHPND) via promoting LDs accumulation and decreasing ROS production.

A transcriptomic map of EGFR-induced epithelial-to-mesenchymal transition identifies prognostic and therapeutic targets for head and neck cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is both a driver oncogene and a therapeutic target in advanced head and neck squamous cell carcinoma (HNSCC). However, response to EGFR treatment is inconsistent and lacks markers for treatment prediction. This study investigated EGFR-induced epithelial-to-mesenchymal transition (EMT) as a central parameter in tumor progression and identified novel prognostic and therapeutic targets, and a candidate predictive marker for EGFR therapy response. METHODS: Transcriptomic profiles were analyzed by RNA sequencing (RNA-seq) following EGFR-mediated EMT in responsive human HNSCC cell lines. Exclusive genes were extracted via differentially expressed genes (DEGs) and a risk score was determined through forward feature selection and Cox regression models in HNSCC cohorts. Functional characterization of selected prognostic genes was conducted in 2D and 3D cellular models, and findings were validated by immunohistochemistry in primary HNSCC. RESULTS: An EGFR-mediated EMT gene signature composed of n = 171 genes was identified in responsive cell lines and transferred to the TCGA-HNSCC cohort. A 5-gene risk score comprising DDIT4, FADD, ITGB4, NCEH1, and TIMP1 prognosticated overall survival (OS) in TCGA and was confirmed in independent HNSCC cohorts. The EGFR-mediated EMT signature was distinct from EMT hallmark and partial EMT (pEMT) meta-programs with a differing enrichment pattern in single malignant cells. Molecular characterization showed that ITGB4 was upregulated in primary tumors and metastases compared to normal mucosa and correlated with EGFR/MAPK activity in tumor bulk and single malignant cells. Preferential localization of ITGB4 together with its ligand laminin 5 at tumor-stroma interfaces correlated with increased tumor budding in primary HNSCC tissue sections. In vitro, ITGB4 knock-down reduced EGFR-mediated migration and invasion and ITGB4-antagonizing antibody ASC8 impaired 2D and 3D invasion. Furthermore, a logistic regression model defined ITGB4 as a predictive marker of progression-free survival in response to Cetuximab in recurrent metastatic HNSCC patients. CONCLUSIONS: EGFR-mediated EMT conveyed through MAPK activation contributes to HNSCC progression upon induction of migration and invasion. A 5-gene risk score based on a novel EGFR-mediated EMT signature prognosticated survival of HNSCC patients and determined ITGB4 as potential therapeutic and predictive target in patients with strong EGFR-mediated EMT.

Comparative Transcriptome Analysis Reveals That WSSV IE1 Protein Plays a Crucial Role in DNA Replication Control.

For DNA viruses, the immediate-early (IE) proteins are generally essential regulators that manipulate the host machinery to support viral replication. Recently, IE1, an IE protein encoded by white spot syndrome virus (WSSV), has been demonstrated to function as a transcription factor. However, the target genes of IE1 during viral infection remain poorly understood. Here, we explored the host target genes of IE1 using RNAi coupled with transcriptome sequencing analysis. A total of 429 differentially expressed genes (DEGs) were identified from penaeid shrimp, of which 284 genes were upregulated and 145 genes were downregulated after IE1 knockdown. GO and KEGG pathway enrichment analysis revealed the identified DEGs are significantly enriched in the minichromosome maintenance (MCM) complex and DNA replication, indicating that IE1 plays a critical role in DNA replication control. In addition, it was found that Penaeus vannamei MCM complex genes were remarkably upregulated after WSSV infection, while RNAi-mediated knockdown of PvMCM2 reduced the expression of viral genes and viral loads at the early infection stage. Finally, we demonstrated that overexpression of IE1 promoted the expression of MCM complex genes as well as cellular DNA synthesis in insect High-Five cells. Collectively, our current data suggest that the WSSV IE1 protein is a viral effector that modulates the host DNA replication machinery for viral replication.

Formation of artificial chromosomes in Caenorhabditis elegans and analyses of their segregation in mitosis, DNA sequence composition and holocentromere organization.

To investigate how exogenous DNA concatemerizes to form episomal artificial chromosomes (ACs), acquire equal segregation ability and maintain stable holocentromeres, we injected DNA sequences with different features, including sequences that are repetitive or complex, and sequences with different AT-contents, into the gonad of Caenorhabditis elegans to form ACs in embryos, and monitored AC mitotic segregation. We demonstrated that AT-poor sequences (26% AT-content) delayed the acquisition of segregation competency of newly formed ACs. We also co-injected fragmented Saccharomyces cerevisiae genomic DNA, differentially expressed fluorescent markers and ubiquitously expressed selectable marker to construct a less repetitive, more complex AC. We sequenced the whole genome of a strain which propagates this AC through multiple generations, and de novo assembled the AC sequences. We discovered CENP-AHCP-3 domains/peaks are distributed along the AC, as in endogenous chromosomes, suggesting a holocentric architecture. We found that CENP-AHCP-3 binds to the unexpressed marker genes and many fragmented yeast sequences, but is excluded in the yeast extremely high-AT-content centromeric and mitochondrial DNA (> 83% AT-content) on the AC. We identified A-rich motifs in CENP-AHCP-3 domains/peaks on the AC and on endogenous chromosomes, which have some similarity with each other and similarity to some non-germline transcription factor binding sites.

RbAp46/48LIN-53 and HAT-1 are required for initial CENP-AHCP-3 deposition and de novo holocentromere formation on artificial chromosomes in Caenorhabditis elegans embryos.

Foreign DNA microinjected into the Caenorhabditis elegans syncytial gonad forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Short, linear DNA fragments injected concatemerize into high molecular weight (HMW) DNA arrays that are visible as punctate DAPI-stained foci in oocytes, and they undergo chromatinization and centromerization in embryos. The inner centromere, inner kinetochore and spindle checkpoint components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, respectively, assemble onto the nascent ACs during the first mitosis. The DNA replication efficiency of ACs improves over several cell cycles, which correlates with the improvement of kinetochore bi-orientation and proper segregation of ACs. Depletion of condensin II subunits, like CAPG-2 and SMC-4, but not the replicative helicase component, MCM-2, reduces de novo CENP-AHCP-3 level on nascent ACs. Furthermore, H3K9ac, H4K5ac and H4K12ac are highly enriched on newly chromatinized ACs. RbAp46/48LIN-53 and HAT-1, which affect the acetylation of histone H3 and H4, are essential for chromatinization, de novo centromere formation and segregation competency of nascent ACs. RbAp46/48LIN-53 or HAT-1 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs. This phenomenon is different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53.

Prognostic models for 1-year survival of NPC after radiotherapy in different ages.

PURPOSE: Previous studies have shown that approximately 10% of nasopharyngeal cancer (NPC) patients die within a year of disease onset, and that age is an independent predictor. However, no predictive model has been developed. We aimed to establish novel prognostic models to predict the 1-year cancer-specific survival (CSS) of young, middle-aged, and older patients with NPC after radiotherapy. METHODS: The data of 2822 NPC patients who underwent radiotherapy between 2004 and 2015 were reviewed from the surveillance, epidemiology, and end results database. We divided them into young, middle-aged, and older people groups according to age (< 44 years, 45-59 years, and ≥ 60 years, respectively). Multivariate analyses were performed, and prognostic models were constructed. RESULTS: Multivariate analyses indicated that age, ethnicity, histological subtype, T, and M stage were independent predictors of 1-year CSS in the older people group. In contrast, ethnicity and age were not found to have predictive value in the young and middle-aged groups, respectively. Accordingly, three prognostic models with excellent predictive values were established for the three groups (C-indices: 0.791 [95% CI 0.722-0.859], 0.763 [95% CI 0.721-0.806] and 0.723 [95% CI 0.683-0.763], respectively). These predictive values are higher than those of the eighth edition American joint committee cancer tumor-node-metastasis (TNM) classification system. CONCLUSION: Three prognostic models for predicting the 1-year CSS of young, middle-aged, and older NPC patients after radiotherapy showed better predictive power than the TNM classification system. These models may guide treatment strategies and clinical decision-making in different cohorts.

Long-term survival trend after primary total laryngectomy for patients with locally advanced laryngeal carcinoma.

Purpose: To evaluate long-term survival trends after primary total laryngectomy (TL) for locally advanced laryngeal carcinoma (LC). Methods: A total of 2094 patients diagnosed with locally advanced LC and underwent primary TL (1992-2011, at least 5-year follow-up) in the Surveillance, Epidemiology, and End Results (SEER) database were included in this study. Besides the traditional overall survival (OS) and cancer-specific survival (CSS) by using Kaplan-Meier curves, the 3-year conditional survival analysis was also performed to describe the long-term trends in these patients. Time-dependent multivariate competing-risk models were constructed to assess the persistent sub-distribution hazard of prognostic factors. Finally, a nomogram was developed to predict conditional cancer-specific survival. Results: The curves of overall hazard and cancer-specific hazard both quickly reached the apex within the first year since TL, then decreased thereafter. In general, the CS3 steadily increased from within 5 years after TL. In the stratified CS3 analysis, the increments in patients with adverse characteristics were more pronounced. 4 years after TL, the probability of surviving the next year exceeded 90%. The time-dependent multivariate competing-risk models indicated that age and lymph node ratio (LNR) persistently contributed to the cancer-specific outcome. The nomogram based on the competing-risk model was constructed to estimate CSS probability conditional upon 3 years for advanced LC patients having survived 1, 2, and 3 years. Conclusion: Most patients achieved a substantially improved survival rate after surviving a long period after primary TL. Patients diagnosed at older age and with higher LNR should receive more effective follow-up. The predictive nomogram can provide significant evidence for clinical research and practice.

Construction and analysis of artificial chromosomes with de novo holocentromeres in Caenorhabditis elegans.

Artificial chromosomes (ACs), generated in yeast (YACs) and human cells (HACs), have facilitated our understanding of the trans-acting proteins, cis-acting elements, such as the centromere, and epigenetic environments that are necessary to maintain chromosome stability. The centromere is the unique chromosomal region that assembles the kinetochore and connects to microtubules to orchestrate chromosome movement during cell division. While monocentromeres are the most commonly characterized centromere organization found in studied organisms, diffused holocentromeres along the chromosome length are observed in some plants, insects and nematodes. Based on the well-established DNA microinjection method in holocentric Caenorhabditis elegans, concatemerization of foreign DNA can efficiently generate megabase-sized extrachromosomal arrays (Exs), or worm ACs (WACs), for analyzing the mechanisms of WAC formation, de novo centromere formation, and segregation through mitosis and meiosis. This review summarizes the structural, size and stability characteristics of WACs. Incorporating LacO repeats in WACs and expressing LacI::GFP allows real-time tracking of newly formed WACs in vivo, whereas expressing LacI::GFP-chromatin modifier fusions can specifically adjust the chromatin environment of WACs. The WACs mature from passive transmission to autonomous segregation by establishing a holocentromere efficiently in a few cell cycles. Importantly, WAC formation does not require any C. elegans genomic DNA sequence. Thus, DNA substrates injected can be changed to evaluate the effects of DNA sequence and structure in WAC segregation. By injecting a complex mixture of DNA, a less repetitive WAC can be generated and propagated in successive generations for DNA sequencing and analysis of the established holocentromere on the WAC.

The primary tumor resection in patients with distant metastatic laryngeal carcinoma.

BACKGROUND: The role of primary tumor resection in patients with distant metastatic laryngeal carcinoma (DMLC) has not been clarified completely. Thus, we used propensity score matching (PSM) and survival analysis to address this issue. METHODS: The PSM was utilized to avoid selection bias and disproportionate distributions of the confounding factors. Kaplan-Meier estimates and Cox proportional hazard analysis were utilized to evaluate overall survival (OS) and cancer-specific survival (CSS). RESULTS: From the Surveillance, Epidemiology, and End Results Program database, a cohort of 480 patients with DMLC were included. After PSM, the OS and CSS for patients who underwent resection were significantly longer than those without resection (median OS: 19 months vs. 8 months, P < 0.001; median CSS: 19 months vs. 9 months, P = 0.002). Tumor resection significantly prolonged survival of DMLC patients with appropriate demographic and clinical characteristics. In the multivariate analysis, age at diagnosis, race, pathologic subtype, and marital status were found significantly affecting both OS and CSS of patients who underwent surgical resection. Predictive nomograms were developed to help distinguish patients with early mortality potential after surgical resection. CONCLUSIONS: This study is the first one using PSM to assess the role played by surgical resection in DMLC and evaluate the prognostic factor of resected patients. Premised on well controlled postoperative complications, resection could significantly prolong OS and CSS of certain patients.

Dynamic prediction of cancer-specific survival for primary hypopharyngeal squamous cell carcinoma.

OBJECTIVES: This study investigated a large cohort of patients to construct a predictive nomogram and a web-based survival rate calculator for dynamically predicting the cancer-specific survival of patients with primary hypopharyngeal squamous cell carcinoma (HSCC). METHODS: Patients (n = 2007) initially diagnosed with primary HSCC from 2004 to 2015 were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. All patients were randomly divided into the training and validation cohorts (1:1). The Lasso Cox regression model was applied to identify independent risk factors of cancer-specific survival for a predictive nomogram and a web-based calculator. The model was evaluated by concordance index, calibration, and decision curve analysis. RESULTS: Cancer-specific survival rates decreased with time, while 3-year conditional survival increased. Cancer-specific deaths evolved from relatively high within the first 3 years to low thereafter. Age, race, T stage, N stage, M stage, surgery, radiotherapy, chemotherapy, and marital status were identified as independent risk factors. We constructed a predictive nomogram for survival and a web-based calculator ( https://linzhongyang.shinyapps.io/Hypopharyngeal/ ). Additionally, a prognostic risk stratification was developed according to nomogram total points. CONCLUSIONS: Patients with primary HSCC were found at a high risk of cancer-specific death during the first 3 years, indicating that additional effective follow-up strategies should be implemented over the period. This is the first study to construct a predictive nomogram and a web-based calculator for all patients with HSCC.

Genetic and epigenetic effects on centromere establishment.

Endogenous chromosomes contain centromeres to direct equal chromosomal segregation in mitosis and meiosis. The location and function of existing centromeres is usually maintained through cell cycles and generations. Recent studies have investigated how the centromere-specific histone H3 variant CENP-A is assembled and replenished after DNA replication to epigenetically propagate the centromere identity. However, existing centromeres occasionally become inactivated, with or without change in underlying DNA sequences, or lost after chromosomal rearrangements, resulting in acentric chromosomes. New centromeres, known as neocentromeres, may form on ectopic, non-centromeric chromosomal regions to rescue acentric chromosomes from being lost, or form dicentric chromosomes if the original centromere is still active. In addition, de novo centromeres can form after chromatinization of purified DNA that is exogenously introduced into cells. Here, we review the phenomena of naturally occurring and experimentally induced new centromeres and summarize the genetic (DNA sequence) and epigenetic features of these new centromeres. We compare the characteristics of new and native centromeres to understand whether there are different requirements for centromere establishment and propagation. Based on our understanding of the mechanisms of new centromere formation, we discuss the perspectives of developing more stably segregating human artificial chromosomes to facilitate gene delivery in therapeutics and research.

A survey of parabens in commercial pharmaceuticals from China and its implications for human exposure.

Parabens are widely used as antimicrobial preservatives during pharmaceutical production. However, little information is available regarding the occurrence of parabens in commercial pharmaceuticals and their implications for human exposure. In this study, six commonly used parabens were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry with 100 commercial pharmaceuticals collected from China. Almost all of the pharmaceutical samples contained at least one kind of parabens with the detection frequency of 97%. The concentrations of Σ6parabens (sum of the six parabens) ranged from below MDL to 1256ng/g, with mean and median values of 94.8 and 119ng/g, respectively. Methyl paraben (MeP), ethyl paraben (EtP) and propyl paraben (PrP) were the predominant compounds. Significant positive correlation was observed between concentrations of MeP and PrP, indicating their co-applications in pharmaceuticals. Levels of Σ6parabens varied in different categories of pharmaceuticals and increased with their shelf lives. Based on the measured concentrations and daily ingestion rates of pharmaceuticals, the estimated daily intake (EDI) of parabens was calculated. The median values of EDIpharmaceutical for male adults, female adults and children were 4.05, 4.75 and 9.73ng/kg-bw/day, respectively, which were three orders of magnitude lower than those from foodstuffs and personal care products (PCPs). It was firstly reported that the total exposure dose was 0.326mg/kg-bw/day via foodstuffs, PCPs, and pharmaceuticals for Chinese female adults.