Engineering Streptomyces albulus to enhance ε-poly-L-lysine production by introducing a polyphosphate kinase-mediated ATP regeneration system.

BACKGROUND: ε-Poly-L-lysine (ε-PL) is a natural and safe food preservative that is mainly produced by filamentous and aerobic bacteria Streptomyces albulus. During ε-PL biosynthesis, a large amount of ATP is used for the polymerization of L-lysine. A shortage of intracellular ATP is one of the major factors limiting the increase in ε-PL production. In previous studies, researchers have mainly tried to increase the oxygen supply to enhance intracellular ATP levels to improve ε-PL production, which can be achieved through the use of two-stage dissolved oxygen control, oxygen carriers, heterologous expression of hemoglobin, and supplementation with auxiliary energy substrates. However, the enhancement of the intracellular ATP supply by constructing an ATP regeneration system has not yet been considered. RESULTS: In this study, a polyphosphate kinase (PPK)-mediated ATP regeneration system was developed and introduced into S. albulus to successfully improve ε-PL production. First, polyP:AMP phosphotransferase (PAP) from Acinetobacter johnsonii was selected for catalyzing the conversion of AMP into ADP through an in vivo test. Moreover, three PPKs from different microbes were compared by in vitro and in vivo studies with respect to catalytic activity and polyphosphate (polyP) preference, and PPK2Bcg from Corynebacterium glutamicum was used for catalyzing the conversion of ADP into ATP. As a result, a recombinant strain PL05 carrying coexpressed pap and ppk2Bcg for catalyzing the conversion of AMP into ATP was constructed. ε-PL production of 2.34 g/L was achieved in shake-flask fermentation, which was an increase of 21.24% compared with S. albulus WG608; intracellular ATP was also increased by 71.56%. In addition, we attempted to develop a dynamic ATP regulation route, but the result was not as expected. Finally, the conditions of polyP6 addition were optimized in batch and fed-batch fermentations, and the maximum ε-PL production of strain PL05 in a 5-L fermenter was 59.25 g/L by fed-batch fermentation, which is the highest ε-PL production reported in genetically engineered strains. CONCLUSIONS: In this study, we proposed and developed a PPK-mediated ATP regeneration system in S. albulus for the first time and significantly enhanced ε-PL production. The study provides an efficient approach to improve the production of not only ε-PL but also other ATP-driven metabolites.

Delayed Unilateral Eagle Syndrome with Fractured Styloid Process.

PURPOSE: Minor injury to head and neck is usually neglected for potential neurological consequences. We report a woman who suffered left Eagle syndrome due to styloid process fracture two years after a minor motorcycle collision. CASE REPORT: A 53-year-old woman complained pain at her left upper neck, lower face and periauricular area after minor motorcycle collision at 2 years ago. The pain usually occurred spontaneously but was occasionally triggered or exacerbated by specific postural changes, including looking up or turning head to right side. Moreover, a foreign body sensation occurred at throat during swallowing. Physical examination provoked pain at the left submandibular area. Carotid bruit was absent. Otherwise, other neurological function was preserved. Computerized tomography revealed linear fracture at the middle of left styloid process. After inform, patient preferred conservative treatments including abortive non- steroidal anti-inflammatory drugs and an avoidance of rapid head rotations. Since afterwards, the frequency and intensity of neck pain greatly decreased and she could tolerate and maintain a normal daily living. CONCLUSIONS: Asymptomatic or paucisymptomatic styloid process fracture may be neglected in case of minor injury to head and neck. A careful evaluation of neck should be completed in traumatic individuals to reveal underlying damage and prevent further harmful consequence.

Prominent changes in blood coagulation of patients with SARS-CoV-2 infection.

Background As the number of patients increases, there is a growing understanding of the form of pneumonia sustained by the 2019 novel coronavirus (SARS-CoV-2), which has caused an outbreak in China. Up to now, clinical features and treatment of patients infected with SARS-CoV-2 have been reported in detail. However, the relationship between SARS-CoV-2 and coagulation has been scarcely addressed. Our aim is to investigate the blood coagulation function of patients with SARS-CoV-2 infection. Methods In our study, 94 patients with confirmed SARS-CoV-2 infection were admitted in Renmin Hospital of Wuhan University. We prospectively collect blood coagulation data in these patients and in 40 healthy controls during the same period. Results Antithrombin values in patients were lower than that in the control group (p < 0.001). The values of D-dimer, fibrin/fibrinogen degradation products (FDP), and fibrinogen (FIB) in all SARS-CoV-2 cases were substantially higher than those in healthy controls. Moreover, D-dimer and FDP values in patients with severe SARS-CoV-2 infection were higher than those in patients with milder forms. Compared with healthy controls, prothrombin time activity (PT-act) was lower in SARS-CoV-2 patients. Thrombin time in critical SARS-CoV-2 patients was also shorter than that in controls. Conclusions The coagulation function in patients with SARS-CoV-2 is significantly deranged compared with healthy people, but monitoring D-dimer and FDP values may be helpful for the early identification of severe cases.

Analysis of Chemical Constituents of Miao Ethnomedicine Heiguteng Zhuifeng Huoluo Capsule (HZFC) and the Discovery of Active Substances in the Treatment of Rheumatoid Arthritis.

In this study, the chemical substances of Heiguteng Zhuifeng Huoluo Capsule (HZFC) and its potential active ingredients for the treatment of rheumatoid arthritis (RA) were characterized and analyzed by medicinal chemistry combined with bioinformatics methods. Also, the potential active ingredients of HZFC against RA were verified by lipopolysaccharide (LPS)-induced macrophage activation model. The results showed that 79 chemical constituents were successfully identified, mainly including phenylpropanoids, flavonoids, and alkaloids. Among them, 13 active components were closely related to the nine core targets (FASN, ALOX5, EGFR, MMP1, CYP2D6, CNR1, AR, MAOA, and FKBP5) of HZFC in the treatment of RA. Molecular docking further proved that 13 active components had strong docking activity with 9 core targets. In the verification experiment of the LPS-induced RAW 264.7 macrophage model, the verified components (magnoflorine, N-feruloyltyramine, canadine, rutin, quercetin-3-O-glucoside, and pseudocolumbamine) all showed a clear inhibitory effect on the secretion of inflammatory factors in model cells. The above research results suggest that 13 components such as stepharanine, rutin, quercetin-3-O-glucoside, corydine methyl ether, canadine, 8-oxoepiberberine, disinomenine, deosinomenine glucoside, tuduranine, magnoflorine, isosinomenine, pseudocolumbamine, and N-feruloyltyramine may be the main active substances of HZFC in the treatment of RA.

Identification of diagnostic biomarkers of rheumatoid arthritis based on machine learning-assisted comprehensive bioinformatics and its correlation with immune cells.

Background: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by inflammatory cell infiltration, which can lead to chronic disability, joint destruction and loss of function. At present, the pathogenesis of RA is still unclear. The purpose of this study is to explore the potential biomarkers and immune molecular mechanisms of rheumatoid arthritis through machine learning-assisted bioinformatics analysis, in order to provide reference for the early diagnosis and treatment of RA disease. Methods: RA gene chips were screened from the public gene GEO database, and batch correction of different groups of RA gene chips was performed using Strawberry Perl. DEGs were obtained using the limma package of R software, and functional enrichment analysis such as gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), disease ontology (DO), and gene set (GSEA) were performed. Three machine learning methods, least absolute shrinkage and selection operator regression (LASSO), support vector machine recursive feature elimination (SVM-RFE) and random forest tree (Random Forest), were used to identify potential biomarkers of RA. The validation group data set was used to verify and further confirm its expression and diagnostic value. In addition, CIBERSORT algorithm was used to evaluate the infiltration of immune cells in RA and control samples, and the correlation between confirmed RA diagnostic biomarkers and immune cells was analyzed. Results: Through feature screening, 79 key DEGs were obtained, mainly involving virus response, Parkinson's pathway, dermatitis and cell junction components. A total of 29 hub genes were screened by LASSO regression, 34 hub genes were screened by SVM-RFE, and 39 hub genes were screened by Random Forest. Combined with the three algorithms, a total of 12 hub genes were obtained. Through the expression and diagnostic value verification in the validation group data set, 7 genes that can be used as diagnostic biomarkers for RA were preliminarily confirmed. At the same time, the correlation analysis of immune cells found that γδT cells, CD4+ memory activated T cells, activated dendritic cells and other immune cells were positively correlated with multiple RA diagnostic biomarkers, CD4+ naive T cells, regulatory T cells and other immune cells were negatively correlated with multiple RA diagnostic biomarkers. Conclusions: The results of novel characteristic gene analysis of RA showed that KYNU, EVI2A, CD52, C1QB, BATF, AIM2 and NDC80 had good diagnostic and clinical value for the diagnosis of RA, and were closely related to immune cells. Therefore, these seven DEGs may become new diagnostic markers and immunotherapy markers for RA.

The clinical significance and potential molecular mechanism of integrin subunit beta 4 in laryngeal squamous cell carcinoma.

The relationship between integrin beta 4 (ITGB4) expression and laryngeal squamous cell carcinoma (LSCC) remains unclarified. The object of the present study was to explore the clinical significance and potential molecular mechanism of ITGB4 in LSCC. The protein level of ITGB4 was significantly higher in 46 LSCC patients than in 26 non-LSCC tissues detected by in-house immunohistochemistry. Consistently, ITGB4 mRNA level was also greatly upregulated based on microarray and RNA-seq data (standard mean difference, SMD = 1.62, 95 % CI: 1.23-2.00). And the area under curves (AUC) of summary receiver operator characteristic (SROC) was 0.87 (95 % CI: 0.84-0.90) based on 172 cases of LSCC and 59 cases of non-cancerous controls. Ninety genes were intersected by the ITGB4 related genes and LSCC differential expressed genes (DEGs) from all available microarray and RNA-seq datasets. Based on Gene Ontology (GO) analysis, the top terms of biological process (BP), cellular component (CC) and molecular function (MF) for the 90 ITGB4 related DEGs were extracellular matrix organization, basement membrane and extracellular matrix structural constituent, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that ITGB4 related DEGs mainly participated in the pathways of ECM-receptor interaction, Focal adhesion and Small cell lung cancer. Moreover, the Protein-Protein Interaction (PPI) network indicated that ITGA3, ITGA5, ITGB4, MET, LAMA3, and COL4A1 might be the core genes of LSCC development related to ITGB4. In conclusion, high ITGB4 expression may lead to the occurrence and development of LSCC via various signaling pathways.

The clinical significance of apolipoprotein L1 in head and neck squamous cell carcinoma.

Approximately 500,000 new head and neck squamous cell carcinoma (HNSCC) cases are detected every year around the world, and its incidence ranks sixth among all cancer types globally. Among these cases, oral squamous cell carcinoma (OSCC) and laryngeal squamous cell carcinoma (LSCC) are HNSCC subtypes with high incidence rates, especially in China. The present study examines the association between the apolipoprotein L1 (APOL1) mRNA and protein expression and clinical parameters in HNSCC. The two most common types (oral and larynx) of HNSCC were selected for subgroup analyses. Immunohistochemistry (IHC) was used to detect APOL1 protein expression levels in HNSCC clinical specimens. It was demonstrated that APOL1 protein expression in 221 cases of HNSCC was higher compared with that in normal tissues. Consistent upregulation of APOL1 protein was also found in subgroups of OSCC and LSCC. Through mining the ArrayExpress, The Cancer Genome Atlas and the Gene Expression Omnibus databases, microarrays and RNA sequencing data for HNSCC were retrieved, which were used to analyze APOL1 mRNA expression levels. The results showed that APOL1 expression was higher in both OSCC and LSCC subtypes, as well as in HNSCC, compared with that in non-cancerous squamous epithelium. The summary receiver operating characteristic analysis showed that APOL1 had potential as a diagnostic biomarker for HNSCC, OSCC and LSCC. Thus, upregulation of APOL1 may contribute to the tumorigenesis of HNSCC.

[Effects of exogenous nitric oxide on chlorophyll in cadmium-induced tomato seedlings].

Cadmium (Cd) is an important heavy metal pollution, and NO is a bioactive molecule, which was found to participate in the reaction of plant to Cd. Leaves from tomato seedlings pretreated with 100 micromol x L(-1) sodium nitroprusside (SNP, as NO donor) 1 day prior to being treated with 50 micromol x L(-1) Cd for 7 days were used as materials, and chloroplasts were isolated from the leaves to study the effects of NO on the spectroscopic characteristics of chlorophyll. The results of absorption spectra of chloroplasts showed that NO alleviated the effects of Cd on absorption spectra of chloroplast by raising the relative absorbance at 436 nm, 480 nm and 470 nm, which caused lower contents of carotinoid and chlorophyll. Fluorescence emission spectra of chloroplasts indicated that NO alleviated effect of Cd, and the relative absorbance at 686 nm and 734 nm decreased 17% and 10% respectively, while they decreased 33% and 23% respectively in chloroplasts treated with Cd. DCPIP analysis results showed that NO alleviated the inhibition of photosynthetic electron transport by Cd, and consequently the electron transport rate reached the same level of control.

[Application of near-infrared spectroscopy to quality detection of milk and its products].

Milk and its products as a kind of ideal comprehensive nutritional food, has becoming an indispensable part of people's daily, life. But at the same time, the quality of dairy products has been also increasingly concerned by consumers. Real-time, rapid and accurate detection of milk and its products in terms of component, adulterants, residues and preservatives is the primary condition for improving the dairy products quality and controlling the production process. Quality predication of milk and its products was often completed by laboratory analysis in the past, which was complicated and time-consuming and could not satisfy the needs for evaluating the milk products quality and monitoring the production proceeding effectively. How to predict the quality of milk and its products quickly and accurately is a practical problem that needs to be resolved. Near-infrared spectroscopy (NIRS) is a rapid, convenient, highly efficient, non-destructive and low-cost analytical technique, which has been widely used in various fields for quantitative and qualitative analysis. As a new analysis technique, NIRS has great potential of application to milk and its products detection, owning to its quick, concise and non--destructive characteristics. The main nutrient components were the major index of milk and its products quality evaluation. Determining the main nutrient components of milk and its products rapidly can provide sound basis for evaluating the products quality. At the same time, adulterants, residues and preservatives were also distinct fingerprint characteristics in the NIR spectra just like the main nutrient components. So this new approaches could also be used in quality distinguishing and on-line detection of milk and its products. Many researches have also concluded that NIRS technology has good stability and high prediction ability on dairy products analysis, exhibites well correlation with the result by labor analysis method. In the present paper, the principles and advantages of NIRS were described. The research advancement of NIRS utilization for milk products nutrient component determination, quality estimation and on-line detection and the application prospect were comprehensively reviewed. With the development of spectral technique, the prediction model gained through NIRS will be more and more reliable and practicable, and the NIRS technique will be more widely used in milk and its products determination, quality estimation and on-line detection.

An accurate quantitative method for screening effective siRNA probes targeting a Hepatitis B virus transcript in single living cells.

A dual fluorescence reporter plasmid expressing EGFP and DsRed-Monomer from separate promoters was constructed for quantitative flow cytometry analysis. Cloning the hepatitis B virus (HBV) X gene into the 3' UTR region of DsRed-Monomer allowed quantifying the efficacy of ten siRNAs designed according to the accessibility of HBx mRNA measured in vitro. Using EGFP as an internal control, a justified calculation of the changed mean fluorescence intensity of DsRed-Monomer in each transfected cell yielded highly consistent results, and revealed all 10 siRNAs achieved over 50% inhibition among which a super effective siRNA achieved 88% inhibition at a very low concentration (0.33 microg/ml). This provides a quantification method critical for therapeutic application of siRNA.

Rapid inactivation of chloroplastic ascorbate peroxidase is responsible for oxidative modification to Rubisco in tomato (Lycopersicon esculentum) under cadmium stress.

To investigate the sensitive site of antioxidant systems in chloroplast under cadmium stress and its consequence on reactive oxygen species production and action, the sub-organellar localization of chloroplast superoxide dismutases (SOD, EC 1.15.1.1) and ascorbic peroxidase (APX, EC 1.11.1.11) isoenzymes and changes of enzymes activities under cadmium stress were investigated in tomato seedlings. Two APX isoforms, one thylakoid-bound and one stromal, were detected. Cd at 50 microM induced a moderate increase of SOD activities but a rapid inactivation of both APX isoenzymes. APX inactivation was mainly related to the decrease of ascorbate concentration, as supported by in vitro treatment of exogenous ascorbate and APX kinetic properties under Cd stress. H2O2 accumulation in chloroplast, as a consequence of APX inactivation, was associated with a 60% loss of Rubisco (EC 4.1.1.39) activity, which could be partially accounted for by a 10% loss of Rubisco content. Protein oxidation assay found that the Rubisco large subunit was the most prominent carbonylated protein; the level of carbonylated Rubisco large subunit increased fivefold after Cd exposure. Thiol groups in the Rubisco large subunit were oxidized, as indicated by non-reducing electrophoresis. Treating crude extract with H2O2 resulted in a similar pattern of protein oxidation and thiols oxidation with that observed in Cd-treated plants. Our study indicates that APXs in the chloroplast is a highly sensitive site of antioxidant systems under Cd stress, and the inactivation of APX could be mainly responsible for oxidative modification to Rubisco and subsequent decrease in its activity.

[Distribution of Pb and Zn in transgenic metallothionein tobacco].

Transgenic metallothionein (MT) plant can clear the heavy metals from soil and environment, but the distribution of metals in plants has not been studied systematically. The Pb and Zn contents in different parts of transgenic MT tobacco plant of sixth generation and traditional plant (same culture variety as control) were analyzed. The Pb and Zn contents in total transgenic plant were 21.8% and 27.2% higher than control, respectively. The distribution of Pb and Zn in different organs varied in these two types of plants. The Pb and Zn contents in old leaves, stem and root in transgenic plants were significantly higher than those in wild type tobacco, while there was no significant difference in young leaves. The Pb contents in old leaves and root were 30.2% and 47.8% higher than those in the control, and the Zn contents in old leaves, stem and root were 4.7%, 29.2% and 21.6% higher than those in the control. These data showed that Pb was accumulated in old leaves and root easily, while Zn was accumulated in old leaves and stem easily.

Inhibition of Hepatitis B virus gene expression by single and dual small interfering RNA treatment.

RNA interference (RNAi) has been successfully applied in suppression of Hepatitis B virus (HBV) replication. To circumvent the problem that mutation in HBV genome may result in resistance when siRNA is further developed as an anti-viral drug, in this study, we established a dual small interfering RNA (siRNA) expression system, which could simultaneously express two different siRNA molecules that can specifically target two genes. To test the effectiveness of this system, we applied this new approach to express simultaneously two different 21-bp hairpin siRNA duplexes that specifically attack the HBs and HBx genes of HBV, respectively, in Bel-7402 and HepG2.2.15 cells. Results indicated that dual siRNA could simultaneously inhibit the expression of HBs and HBx gene by 83.7% and 87.5%, respectively, based on luciferase assays. In addition, dual siRNA molecules were able to significantly reduce the amount of HBV core associated DNA, which is considered as an intracellular replicative intermediate, and the viral DNA in culture supernatant. Therefore, this dual siRNA system provides a more powerful tool for the study of gene function and implicates a potential application in the treatment of viral infection.

[RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells].

OBJECTIVES: To investigate the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells. METHODS: The four siRNA against MAT 2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6. We inserted the target sequence of MAT 2A gene into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucA-MAT 2A. The recombinant plasmid and siRNA-producing plasmid were co-transfected into 293 T cells using this construct via lipofectamine methods. The inhibition effect was detected by measuring luciferase activity in the cell lysate to screen the effective siRNA, and then, the effective siRNA was transfected into Bel-7402 cells. The effect of siRNA treatment on the MAT 2A mRNA level and the MAT activity of hepatoma cells were measured. In order to study the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells, the tumor cell killing rate was analyzed by MTT method and the rate of apoptosis of hepatoma cells was evaluated by flow cytometry. RESULTS: The two siRNA among the four siRNA displayed inhibitory effect on the lucifermase expression with the inhibitory rates of 81% and 89% respectively. The expression of MAT 2A mRNA in Bel-7402 cells was specifically inhibited and the MAT activity in Bel-7402 cells was decreased. Furthermore, silencing of the MAT 2A gene by RNAi significantly inhibited hepatoma cell growth and led to induction of apoptosis. CONCLUSION: RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells; MAT 2A is an ideal target of gene-specific therapy for liver cancer.

Hepatitis B virus upregulates the expression of kinesin family member 4A.

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). Kinesin family member 4A (KIF4A) is a microtubule­based motor protein, which is upregulated in cervical and lung cancer. However, the expression of KIF4A in HBV­associated HCC, and the effect of HBV on the expression of KIF4A remain to be elucidated. In the present study, the expression profiles of KIF4A were examined in cancerous tissues and paracancerous tissues from patients with HCC, who presented with histories of chronic HBV infection, and the role of HBV in the induction of the expression of KIF4A was investigated. HepG2 cells were transfected with the pHBV1.3, HBV infectious clone and a construct, which contained the luciferase gene under the control of the KIF4A gene promoter. The results demonstrated that the expression of KIF4A was significantly higher in the HCC tissues than in the paracancerous tissues. HBV activated the KIF4A gene promoter and upregulated the mRNA and protein expression of KIF4A. Furthermore, activation of the gene expression of KIF4A increased in a pHBV1.3 concentration­dependent manner. These results provide novel insights into the understanding of HCC oncogenesis caused by HBV.

RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells.</p><p><b>METHODS</b>The four siRNA against MAT 2A gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilence-2.1-U6. We inserted the target sequence of MAT 2A gene into the upstream of the reporter gene in order to construct the recombinant plasmid vector plucA-MAT 2A. The recombinant plasmid and siRNA-producing plasmid were co-transfected into 293 T cells using this construct via lipofectamine methods. The inhibition effect was detected by measuring luciferase activity in the cell lysate to screen the effective siRNA, and then, the effective siRNA was transfected into Bel-7402 cells. The effect of siRNA treatment on the MAT 2A mRNA level and the MAT activity of hepatoma cells were measured. In order to study the effect of short interfering RNA targeting MAT 2A on growth and apoptosis of hepatoma cells, the tumor cell killing rate was analyzed by MTT method and the rate of apoptosis of hepatoma cells was evaluated by flow cytometry.</p><p><b>RESULTS</b>The two siRNA among the four siRNA displayed inhibitory effect on the lucifermase expression with the inhibitory rates of 81% and 89% respectively. The expression of MAT 2A mRNA in Bel-7402 cells was specifically inhibited and the MAT activity in Bel-7402 cells was decreased. Furthermore, silencing of the MAT 2A gene by RNAi significantly inhibited hepatoma cell growth and led to induction of apoptosis.</p><p><b>CONCLUSION</b>RNA interference-mediated silencing of MAT 2A gene attenuates growth and induces apoptosis of hepatoma cells; MAT 2A is an ideal target of gene-specific therapy for liver cancer.</p>