Metabolic engineering of Halomonas bluephagenesis for production of five carbon molecular chemicals derived from L-lysine.

5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB-co-5HV) of 3-hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117ΔgabT1+2, which produced 16.4 g L-1 and 67.4 g L-1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on Pporin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC produced 6 g L-1 5HV in shake flask growth, while H. bluephagenesis YF117 harboring pSEVA341-Pporin42-yqhDEC-Pporin278-phaCRE-abfT synthesized 42 wt% P(3HB-co-4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB-co-5HV) utilizing L-lysine as the substrate.

Flux optimization using multiple promoters in Halomonas bluephagenesis as a model chassis of the next generation industrial biotechnology.

Predictability and robustness are challenges for bioproduction because of the unstable intracellular synthetic activities. With the deeper understanding of the gene expression process, fine-tuning has become a meaningful tool for biosynthesis optimization. This study characterized several gene expression elements and constructed a multiple inducible system that responds to ten different small chemical inducers in halophile bacterium Halomonas bluephagenesis. Genome insertion of regulators was conducted for the purpose of gene cluster stabilization and regulatory plasmid simplification. Additionally, dynamic ranges of the multiple inducible systems were tuned by promoter sequence mutations to achieve diverse scopes for high-resolution gene expression control. The multiple inducible system was successfully employed to precisely control chromoprotein expression, lycopene and poly-3-hydroxybutyrate (PHB) biosynthesis, resulting in colorful bacterial pictures, optimized cell growth, lycopene and PHB accumulation. This study demonstrates a desirable approach for fine-tuning of rational and efficient gene expressions, displaying the significance for metabolic pathway optimization.

A long-term growth stable Halomonas sp. deleted with multiple transposases guided by its metabolic network model Halo-ecGEM.

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.

Dexmedetomidine improves the acute stress reactivity of male rat through interventions of serum- and glucocorticoid-inducible kinase 1 and nNOS in the bed nucleus of the stria terminalis.

Moderate acute stress responses are beneficial for adaptation and maintenance of homeostasis. Exposure of male rat to stress induces effects in the bed nucleus of the stria terminalis (BNST), for it can be activated by the same stimuli that induce activation of the hypothalamic-pituitary-adrenal axis. However, the underlying mechanism of the BNST on male stress reactivity remains unclear. In this study, we explored whether systematic administration of dexmedetomidine (DEXM) altered the acute stress reactivity through its effect on the BNST. Male Sprague-Dawley rats in the stress (STRE) group, DEXM group, and the DEXM + GSK-650394 (GSK, an antagonist of serum- and glucocorticoid-inducible kinase 1 (SGK1)) group, except those in the vehicle (VEH) group, underwent 1-h restraint plus water-immersion (RPWI) exposure. All the rats proceeded the open field test (OFT) 24 h before RPWI and 1 h after RPWI. After the second OFT, the rats received VEH, DEXM (75 µg/kg i.p.), or were pretreated with GSK (2 µM i.p.) 0.5 h ahead of DEXM respectively. The third OFT was conducted 6 h after drug administration and then the rats were sacrificed. The rats that experienced RPWI showed dramatically elevated serum corticosterone (CORT), multiplied neuronal nitric oxide synthase (nNOS) and SGK1 in the BNST, and terrible OFT behavior. We discovered when the nNOS and SGK1 were decreased in the rat BNST through DEXM treatment, the serum CORT was reduced and the OFT manifestation was ameliorated, whereas these were restrained by GSK application. Our results reveal that modest interventions to SGK1 and nNOS in the BNST improve the male rat reactivity to acute stress, and DEXM was one modulator of these effects.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution.

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Engineering an oleic acid-induced system for Halomonas, E. coli and Pseudomonas.

Ligand-induced system plays an important role for microbial engineering due to its tunable gene expression control over timings and levels. An oleic acid (OA)-induced system was recently constructed based on protein FadR, a transcriptional regulator involved in fatty acids metabolism, for metabolic control in Escherichia coli. In this study, we constructed a synthetic FadR-based OA-induced systems in Halomonas bluephagenesis by hybridizing the porin promoter core region and FadR-binding operator (fadO). The dynamic control range was optimized over 150-fold, and expression leakage was significantly reduced by tuning FadR expression and positioning fadO, forming a series of OA-induced systems with various expression strengths, respectively. Additionally, ligand orthogonality and cross-species portability were also studied and showed highly linear correlation among Halomonas spp., Escherichia coli and Pseudomonas spp. Finally, OA-induced systems with medium- and small-dynamic control ranges were employed to dynamically control the expression levels of morphology associated gene minCD, and monomer precursor 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathway for polyhydroxyalkanoates (PHA), respectively, in the presence of oleic acid as an inducer. As a result, over 10 g/L of poly-3-hydroxybutyrate (PHB) accumulated by elongated cell sizes, and 6 g/L of P(3HB-co-9.57 mol% 4HB) were obtained by controlling the dose and induction time of oleic acid only. This study provides a systematic approach for ligand-induced system engineering, and demonstrates an alternative genetic tool for dynamic control of industrial biotechnology.

Reference Module-Based Analysis of Ovarian Cancer Transcriptome Identifies Important Modules and Potential Drugs.

Ovarian cancer (OVC) is often diagnosed at the advanced stage resulting in a poor overall outcome for the patient. The disease mechanisms, prognosis, and treatment require imperative elucidation. A rank-based module-centric framework was proposed to analyze the key modules related to the development, prognosis, and treatment of OVC. The ovarian cancer cell line microarray dataset GSE43765 from the Gene Expression Omnibus database was used to construct the reference modules by weighted gene correlation network analysis. Twenty-three reference modules were tested for stability and functionally annotated. Furthermore, to demonstrate the utility of reference modules, two more OVC datasets were collected, and their gene expression profiles were projected to the reference modules to generate a module-level expression. An epithelial-mesenchymal transition module was activated in OVC compared to the normal epithelium, and a pluripotency module was activated in ovarian cancer stroma compared to ovarian cancer epithelium. Seven differentially expressed modules were identified in OVC compared to the normal ovarian epithelium, with five up-regulated, and two down-regulated. One module was identified to be predictive of patient overall survival. Four modules were enriched with SNP signals. Based on differentially expressed modules and hub genes, five candidate drugs were screened. The hub genes of those modules merit further investigation. We firstly propose the reference module-based analysis of OVC. The utility of the analysis framework can be extended to transcriptome data of other kinds of diseases.

Engineering Halomonas bluephagenesis as a chassis for bioproduction from starch.

Halomonas bluephagenesis has been successfully engineered to produce multiple products under open unsterile conditions utilizing costly glucose as the carbon source. It would be highly interesting to investigate if H. bluephagenesis, a chassis for the Next Generation Industrial Biotechnology (NGIB), can be reconstructed to become an extracellular hydrolytic enzyme producer replacing traditional enzyme producer Bacillus spp. If successful, cost of bulk hydrolytic enzymes such as amylase and protease, can be significantly reduced due to the contamination resistant and robust growth of H. bluephagenesis. This also allows H. bluephagenesis to be able to grow on low cost substrates such as starch. The modularized secretion machinery was constructed and fine-tuned in H. bluephagenesis using codon-optimized gene encoding α-amylase from Bacillus lichenifomis. Screening of suitable signal peptides and linkers based on super-fold green fluorescence protein (sfGFP) for enhanced expression in H. bluephagenesis resulted in a 7-fold enhancement of sfGFP secretion in the recombinant H. bluephagenesis. When the gene encoding sfGFP was replaced by α-amylase encoding gene, recombinant H. bluephagenesis harboring this amylase secretory system was able to produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), ectoine and L-threonine utilizing starch as the growth substrate, respectively. Recombinant H. bluephagenesis TN04 expressing genes encoding α-amylase and glucosidase on chromosome and plasmid-based systems, respectively, was able to grow on corn starch to approximately 10 g/L cell dry weight containing 51% PHB when grown in shake flasks. H. bluephagenesis was demonstrated to be a chassis for productions of extracellular enzymes and multiple products from low cost corn starch.

Network analysis reveals important genes in human placenta.

AIM: To determine which genes are important in placenta by network analysis. METHODS: Placenta expressing genes were screened from RNA-Seq data. Protein-protein interaction data were downloaded from STRING (v11.0) database. Google PageRank (PR) algorithm was used to identify important placental genes from protein interaction network. Six placental disease-related datasets were downloaded from NCBI GEO database, and the differential expression of the 99 genes was identified. RESULTS: We calculated PR for each placenta expressing gene and defined the top 99 genes with high PR as important genes. GAPDH has the highest PR. The 99 genes had different expression pattern in placental cell types. FN1 is up-regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. HSPA4 is down-regulated in 8 w EVT compared to 8 w CTB and 24 w EVT compared to 8 w EVT. MIB2, TLR4, and UBB are consistently changed in preeclampsia (PE). UBB and ACTG1 were identified to be down-regulated in fetal growth restriction (FGR). SOD1 is down-regulated in preterm birth placenta. CONCLUSION: Our findings confirmed that the importance of these genes in placenta-related diseases, and provide new candidates (MIB2, UBB, ACTG1, and SOD1) for placenta-related disease diagnosis and treatment.

MicroRNAs and Transcripts Associated with an Early Ripening Mutant of Pomelo (Citrus grandis Osbeck).

'Liuyuezaoyou' is an early-ripening cultivar selected from a bud mutation of Citrus grandis Osbeck 'Guanximiyou'. They were designated here as MT and WT, respectively. The fruit of MT matures about 45 days earlier than WT, which was accompanied by significant changes in key phytohormones, sugar compounds and organic acids. Recent studies have showed that microRNAs (miRNAs) play an important role in regulation of fruit ripening process. The aim of this study was to compare MT fruits with WT ones to uncover if miRNAs were implicated in the ripening of C. grandis. Fruits of both WT and MT at four developmental stages were analyzed using high-throughput sequencing and RT-PCR. Several independent miRNA libraries were constructed and sequenced. A total of 747 known miRNAs were identified and 99 novel miRNAs were predicted across all libraries. The novel miRNAs were found to have hairpin structures and possess star sequences. These results showed that transcriptome and miRNAs are substantially involved in a complex and comprehensive network in regulation of fruit ripening of this species. Further analysis of the network model revealed intricate interactions of miRNAs with mRNAs during the fleshy fruit ripening process. Several identified miRNAs have potential targets. These include auxin-responsive protein IAA9, sucrose synthase 3, V-type proton ATPase, NCED1 (ABA biosynthesis) and PL1/5 (pectate lyase genes), as well as NAC100 putative coordinated regulation networks, whose interactions with respective miRNAs may contribute significantly to fruit ripening of C. grandis.

Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas.

Optimization of intracellular biosynthesis process involving regulation of multiple gene expressions is dependent on the efficient and accurate expression of each expression unit independently. However, challenges of analyzing intermediate products seriously hinder the application of high throughput assays. This study aimed to develop an engineering approach for unsterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) or (P3HB4HB) by recombinant Halomonas bluephagenesis (H. bluephagenesis) constructed via coupling the design of GFP-mediated transcriptional mapping and high-resolution control of gene expressions (HRCGE), which consists of two inducible systems with high- and low-dynamic ranges employed to search the exquisite transcription level of each expression module in the presence of γ-butyrolactone, the intermediate for 4-hydroxybutyrate (4HB) synthesis. It has been successful to generate a recombinant H. bluephagenesis, namely TD68-194, able to produce over 36 g/L P3HB4HB consisting of 16 mol% 4HB during a 7-L lab-scale fed-batch growth process, of which cell dry weight and PHA content reached up to 48.22 g/L and 74.67%, respectively, in 36 h cultivation. HRCGE has been found useful for metabolic pathway construction.

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.

Oridonin inhibits hypoxia-induced epithelial-mesenchymal transition and cell migration by the hypoxia-inducible factor-1α/matrix metallopeptidase-9 signal pathway in gallbladder cancer.

Hypoxia has crucial roles in cancer development and progression. Our previous study indicated that cell migration was increased in a hypoxic microenvironment in GBC-SD gallbladder cancer (GBC) cells. Oridonin, a bioactive diterpenoid compound that is isolated from the plant Rabdosia rubescens, has been identified as an anticancer agent in various types of cancer. However, its roles in cell proliferation, apoptosis, and migration in a hypoxic microenvironment and the associated regulatory mechanisms have not yet to be fully elucidated in GBC. The present study investigated the effect of oridonin on cell proliferation, apoptosis, the cell cycle and cell migration in GBC in vitro and in vivo. Furthermore, the role of oridonin in hypoxia-induced cell migration and its underlying mechanisms were explored in GBC. The results indicated that treatment with oridonin significantly suppressed cell proliferation and the metastatic ability of GBC-SD cells in a dose-dependent manner, increased the level of cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Further experiments demonstrated that oridonin could inhibit hypoxia-induced epithelial-mesenchymal transition and cell migration by downregulating the expression levels of hypoxia-inducible factor (HIF)-1α/matrix metallopeptidase (MMP)-9. In addition, oridonin suppressed GBC cell growth and downregulated the expression levels of HIF-1α and MMP-9 in a GBC-SD cell xenograft model. Taken together, these results suggest that oridonin possesses anticancer properties in GBC. Notably, oridonin can suppress tumor epithelial-mesenchymal transition and cell migration by targeting the HIF-1α/MMP-9 signaling pathway.

Build-up of a continuous flow pre-coated dynamic membrane filter to treat diluted textile wastewater and identify its dynamic membrane fouling.

This research built up a continuous dynamic flow filter membrane to treat diluted textile wastewater and basically investigated dynamic membrane fouling mechanism. By pre-depositing particles activated carbon (PAC) on membrane support material (MSM), a thin layer was formed on its surface, which showed excellent results in removing organic pollutants from diluted textile wastewater. Experimental data were regressed by the Langmuir, Freundlich, Temkin, Dubinin-Radushkevich (D-R) and Sips isotherm models. The three two-parameter isotherms (Temkin, D-R and Freundlich) were the models that best fitted, with respectively 0.977, 0.975 and 0.973 regression coefficients. D-R model has registered the maximum calculated adsorption capacity Qmd, cal. = 45.499 mg/g and the mean energy which was required to adsorb 1 mol of MB dye by the DM layer E = 4.249 kJ/mol; indicating the energy distribution onto heterogeneous surface of a physical adsorption process. Furthermore, kinetic models results showed that MB adsorption onto PAC at different initial concentrations follows the pseudo-second order. The obtained results also indicated that a flexible DM layer with different thickness can be formed from different amount of PAC pre-deposited on MSMs, which demonstrated that it was convenient to adjust the required DM thickness to filtrate a known initial concentration for >99% organic pollutants removal efficiency rate. However, DM fouling occurred on small pores MSMs; which resulted in an increase of the filtration pressure what have affected the filtration performance. PAC and MSMs surface morphology and texture structure, before and after filtration, were visualized respectively by Field Emission Scanning Electron Microscopy (FESEM), Fourier Transform Infra-Red and Attenuated Total Reflectance (FTIR/ATR). From these experimental results, a sustainable flux (>6.85 × 10-5 m/s) was established to discriminate no fouling from fouling conditions based on flux and TMP trends variance.

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.

Validation of the American Joint Committee on Cancer eighth edition staging system in patients undergoing hepatectomy for hepatocellular carcinoma: a US population-based study.

BACKGROUND: The American Joint Committee on Cancer (AJCC) eighth edition staging system for hepatocellular carcinoma (HCC) has incorporated several significant changes. This study aims to evaluate the newly proposed staging system and assess its strengths and weaknesses. MATERIALS AND METHODS: Using the Surveillance, Epidemiology, and End Results database, we identified patients with pathologically confirmed stage I-III HCC diagnosed between 2004 and 2014. RESULTS: After all exclusion criteria were applied, AJCC seventh and eighth edition staging was possible in 4931 patients. According to the AJCC eighth edition staging system, stages IB and II did not differ significantly in terms of overall survival (OS) and cause-specific survival (CSS) (P = 0.928 and 0.872, respectively). On the basis of the above results, we reclassified T1a, T1b, and T2 into several subgroups. According to the modified AJCC eighth edition staging system, OS and CSS among the five groups of patients differed significantly. For OS predication, the Akaike information criterion values for the AJCC seventh, eighth, and modified eighth edition staging systems were 29,288.24, 29,282.85, and 27,182.21, respectively, and the c-indices for the AJCC seventh, eighth, and modified eighth edition staging systems were 0.5716, 0.5805, and 0.6082, respectively. Regarding CSS, the Akaike information criterion values for the AJCC seventh, eighth, and modified eighth edition staging systems were 21,701.11, 21,682.12, and 20,313.26, respectively, and the c-indices for the AJCC seventh, eighth, and modified eighth edition staging systems were 0.5983, 0.6117, and 0.6436, respectively. CONCLUSIONS: This is the first large-scale validation of the AJCC eighth edition staging system for patients undergoing hepatectomy. Our study revealed that there was a lack of discrepancy in the outcomes for stage IB and II tumors for AJCC eighth edition staging system of HCC. Our modified AJCC eighth edition staging system allows for finer stratification of patients undergoing hepatectomy according to more detailed tumor size and vascular invasion classifications.

[Relationship between the expression of immunoglobulin heavy chain binding protein and calreticulin and metastasis in hepatocellular carcinoma].

OBJECTIVE: To investigate the relationship between the expression of immunoglobulin heavy chain binding protein (Bip) and calreticulin (CRT) and metastasis in hepatocellular carcinoma. METHODS: Western blot and RT-qPCR were used to detect the expression of Bip and CRT in normal hepatic cells and hepatocellular carcinoma cells. 43 cases of hepatocellular carcinoma tissues were collected from the First Affiliated Hospital of Zhengzhou University between June 2014 and May 2015, Western blot and immunohistochemistry assay were used to detect the expression of Bip and CRT in hepatocellular carcinoma tissues, adjacent non-cancer tissues, 5 cases adjacent liver hemangioma tissues and the clinical significance was also analyzed. RESULTS: The expression of Bip and CRT in hepatocellular carcinoma cells and hepatocellular carcinoma tissues was higher than normal hepatic cells and adjacent non-cancer tissues and also adjacent liver hemangioma tissues. The positive rate of Bip and CRT were 86.0%(37/43) and 65.1%(28/43) in 43 hepatocellular carcinoma tissues, which was significantly higher than those adjacent non-cancer tissues and adjacent liver hemangioma tissues (P<0.05). The positive rate of Bip and CRT in metastatic cancer tissues were 100%(19/19), 84.2%(16/19), respectively, which was significantly higher than those non-metastatic cancer tissues 75.0%(18/24), 50.0%(12/24) respectively (P<0.05). The expression of Bip and CRT was positively correlated (r=0.382, P<0.05). The positive rate and relative expression of Bip were correlated with TNM stage, metastasis in hepatocellular carcinoma tissues (P<0.05), while the positive rate and relative expression of CRT were correlated with metastasis in hepatocellular carcinoma tissues (P<0.05). CONCLUSIONS: The expression of Bip and CRT is correlation with metastasis in hepatocellular carcinoma.

Storage-D: A user-friendly platform that enables practical and personalized DNA data storage.

Deoxyribonucleic acid (DNA) has been suggested as a very promising medium for data storage in recent years. Although numerous studies have advocated for DNA data storage, its practical application remains obscure and there is a lack of a user-oriented platform. Here, we developed a DNA data storage platform, named Storage-D, which allows users to convert their data into DNA sequences of any length and vice versa by selecting algorithms, error-correction, random-access, and codec pin strategies in terms of their own choice. It incorporates a newly designed "Wukong" algorithm, which provides over 20 trillion codec pins for data privacy use. This algorithm can also control GC content to the selected standard, as well as adjust the homopolymer run length to a defined level, while maintaining a high coding potential of ~1.98 bis/nt, allowing it to outperform previous algorithms. By connecting to a commercial DNA synthesis and sequencing platform with "Storage-D," we successfully stored "Diagnosis and treatment protocol for COVID-19 patients" into 200 nt oligo pools in vitro, and 500 bp genes in vivo which replicated in both normal and extreme bacteria. Together, this platform allows for practical and personalized DNA data storage, potentially with a wide range of applications.

Effect of seabuckthorn seed protein and its arginine-enriched peptides on combating memory impairment in mice.

The current study characterized the combating memory impairment effect of seabuckthorn seed protein (SSP) and the arginine (Arg)-enriched peptides (SSPP) on d-galactose-induced brain aging in mice. The Arg content in SSP and SSPP were 10.11 and 17.82 g/100 g, respectively. Seven Arg peptides (Ile/Leu-Arg, Arg-Glu, Asp-Arg-Pro, Arg-Try-Ala, Glu-Arg-Ser, Val-Gly-Arg-Pro, and Lys-Thr-Glu-Arg) were identified from SSPP. The animal experiments of the Morris water maze and the step-down test indicated that the oral administration of SSP (0.25, 0.5, 1.0 mg/g·d) and SSPP (0.25, 0.5, 1.0 mg/g·d) significantly (p < 0.05) reversed the learning and memory impairment symptoms. The activation of endothelial nitric oxide (NO) synthase and neuronal NO synthase were increased, and inducible NO synthase decreased after SSP and SSPP in the hippocampus compared to the model group, with the SSPP being quite effective. Moreover, the treatment significantly exhibited the ability to normalize the serum inflammatory cytokine levels (NF-ĸB, TNF-α, IL-6) and suppress the Arg-inducible nitric oxide (Arg-iNO) pathway. Therefore, SSP and SSPP ingestion reversed the behavioral learning and memory impairment symptoms possibly associated with the anti-inflammation and Arg-iNO pathway. Consumption of SSP and SSPP diets can be beneficial to memory impairment.