AlgU mediates hyperosmotic tolerance in Pseudomonas protegens SN15-2 by regulating membrane stability, ROS scavenging, and osmolyte synthesis.

Pseudomonas protegens can serve as an agricultural biocontrol agent. P. protegens often encounters hyperosmotic stress during industrial production and field application. The ability of P. protegens to withstand hyperosmotic stress is important for its application as a biocontrol agent. AlgU is a global regulator responsible for stress response and biocontrol ability. However, the specific regulatory role of AlgU in the hyperosmotic adaptation of P. protegens is poorly understood. In this study, we found that the AlgU mutation disrupted the hyperosmotic tolerance of P. protegens. Many genes and metabolites related to cell envelope formation were significantly downregulated in ΔalgU compared with that in the wild-type (WT) strain under hyperosmotic conditions, and we found that the algU mutation caused membrane integrity to be compromised and increased membrane permeability. Further experiments revealed that the cell envelope integrity protein TolA, which is regulated by AlgU, contributes to cell membrane stability and osmotic tolerance in P. protegens. In addition, several genes related to oxidative stress response were significantly downregulated in ΔalgU, and higher levels of intracellular reactive oxygen species were found in ΔalgU. Furthermore, we found that the synthesis of N-acetyl glutaminyl glutamine amide is directly regulated by AlgU and contributes to the hyperosmotic adaptation of P. protegens. This study revealed the mechanisms of AlgU's participation in osmotic tolerance in P. protegens, and it provides potential molecular targets for research on the hyperosmotic adaptation of P. protegens.IMPORTANCEIn this study, we found that the extracytoplasmic function sigma factor AlgU is essential for the survival of P. protegens under hyperosmotic conditions. We provided evidence supporting the roles of AlgU in influencing cell membrane stability, intracellular reactive oxygen species (ROS) accumulation, and dipeptide N-acetylglutaminylglutamine amide (NAGGN) synthesis in P. protegens under hyperosmotic conditions. Our findings revealed the mechanisms of AlgU's participation in hyperosmotic stress tolerance in P. protegens, and they provide potential molecular targets for research on the hyperosmotic adaptation of P. protegens, which is of value in improving the biocontrol ability of P. protegens.

Ephrin-A4 Ligand (EFNA4) Predicts Poor Prognosis of Hepatocellular Carcinoma and Promotes Tumor Proliferation.

Background/Aims: Primary hepatocellular carcinoma (HCC) is one of the most lethal tumor diseases in the world. Receptor tyrosine kinases (RTKs) are thought to play a vital role in HCC and Ephrin-A4 ligand (EFNA4) is a membrane-bound molecule that can activate RTKs through erythropoietin-producing hepatocellular (Eph) receptors. However, the specific role of EFNA4 remains unknown. The aim of our study was to explore the prognostic value of EFNA4 expression in HCC. Methods: Bioinformatics analyses were conducted to probe the expression levels and prognostic value of EFNA4 in HCC. The quantitative real-time polymerase chain reaction, immunohistochemical and western blot were used to confirm the expression of EFNA4 in paired clinical specimens of HCC. Colony formation assay was used to confirm the proliferation of tumor cell. Results: The expression of EFNA4 is generally elevated in various cancers. Especially, EFNA4 was upregulated in tumor tissue and associated with clinical stage in HCC patients. HCC patients with lower levels of EFNA4 possessed better survival and progression-free survival times. Colony formation assay indicated that the overexpression of EFNA4 promoted tumor cell proliferation. Conclusion: These results demonstrated that EFNA4 played as an oncogenic gene and a prognostic biomarker for HCC patients.

AlgU controls environmental stress adaptation, biofilm formation, motility, pyochelin synthesis and antagonism potential in Pseudomonas protegens SN15-2.

Pseudomonas protegens is a typical plant-growth-promoting rhizobacterium that can serve as an agricultural biocontrol agent. The extracytoplasmic function (ECF) sigma factor AlgU is a global transcription regulator controlling stress adaption and virulence in Pseudomonas aeruginosa and Pseudomonas syringae. Meanwhile, the regulatory role of AlgU in the biocontrol ability of P.protegens has been poorly studied. In this study, deletion mutations of algU and its antagonist coding gene mucA were constructed to investigate the function of AlgU in P.protegens SN15-2 via phenotypic experiment and transcriptome sequencing analysis. On the basis of phenotypic analyses, it was concluded that the AlgU whose transcription was induced by osmotic stress and oxidative stress positively regulated biofilm formation and tolerance towards osmotic, heat, and oxidation stresses, while it negatively regulated motility, pyochelin synthesis, and the ability to inhibit pathogens. On the basis of the RNA-seq analysis, compared to the wild-type strain, 12 genes were significantly upregulated and 77 genes were significantly downregulated in ΔalgU, while 407 genes were significantly upregulated and 279 genes were significantly downregulated in ΔmucA, indicating the involvement of AlgU in several cellular processes, mainly related to resistance, carbohydrate metabolism, membrane formation, alginate production, the type VI secretion system, flagella motility and pyochelin production. Our findings provide insights into the important role of AlgU of P.protegens in biocontrol, which is of value in improving the biocontrol ability of P.protegens.

Detection of Coccidioides posadasii in a patient with meningitis using metagenomic next-generation sequencing: a case report.

BACKGROUND: Coccidioidomycosis is a systemic infection caused by dimorphic fungi Coccidioides spp. endemic to Southwestern United States and Central and South America. A history of residence and travel in these areas is essential for the diagnostic of coccidioidomycosis, which has highly variable symptoms ranging from asymptomatic to severe, disseminated infection, and even death. Immunocompromised patients of coccidioidomycosis experience a high risk of dissemination, chronic infection, and mortality. Meningitis is one of the most deleterious coccidioidomycosis and can cause various life-threatening complications. CASE PRESENTATION: Here we report a case of Coccidioides posadasii meningitis in a 49-year-old female who returned to China after one and a half years residence in Los Angeles, USA. The repeated routine cultures using CSF for bacteria or fungi were all negative. To hunt for an infectious etiology, the state-of-the-art technology metagenomic next-generation sequencing (mNGS) was then utilized, suggesting Coccidioides posadasii. Organizational pathological examination and polymerase-chain-reaction (PCR) results subsequently confirmed the mNGS detection. CONCLUSION: To our knowledge, cases for coccidioidal meningitis have been rarely reported in China. While global travelling may spread this disease across continents and make the diagnosis more difficult. mNGS can detect almost all known pathogens with high sensitivity and specificity, especially for uncommon pathogen, such as Coccidioides posadasii in China.

Hepatitis B core antibody and liver stiffness measurements predict HBeAg seroconversion in HBeAg-positive chronic hepatitis B patients with minimally elevated alanine aminotransferase (ALT) levels.

Alanine aminotransferase (ALT) levels between 1 and 2 times the upper limit of normal (ULN) are common in patients with chronic hepatitis B (CHB) infection. There are few clinical studies focused on this group of patients because of the poorer treatment outcomes compared to those with more than 2 × ULN ALT level. However, treatments are necessary to reduce liver damage for patients with minimally elevated ALT levels. And biomarkers are needed in predicting the treatment response. In this study, a total of 106 patients with CHB were enrolled and treated with entecavir, telbivudine or tenofovir disoproxil fumarate. Liver stiffness was measured by transient elastography, and quantitative levels of hepatitis B core antibody (HBcAb) were detected by ELISA. At week 96, 31 (29.25%) patients achieved hepatitis B e antigen (HBeAg) seroconversion. Notably, baseline HBcAb levels and liver stiffness measurements (LSM) were higher in patients who achieved HBeAg seroconversion. The multivariate analysis showed that the baseline HBcAb levels and LSM were independent predictors for HBeAg seroconversion. The area under receiver operating characteristic curve of baseline HBcAb, LSM and the combination of them for HBeAg seroconversion was 0.714, 0.720 and 0.717, respectively. In addition, we discovered that the patients with baseline HBcAb levels ≥ 4.15 log10 IU/mL and LSM ≥ 9.85 kPa had higher rates of HBeAg seroconversion. Therefore, the measurement of HBcAb and liver stiffness might be good approaches for the optimization of antiviral therapy for HBeAg-positive CHB patients with minimally elevated ALT levels.

Transcriptomic response of Ralstonia solanacearum to antimicrobial Pseudomonas fluorescens SN15-2 metabolites.

To develop efficient biocontrol agents, it is essential to investigate the response of soil-borne plant pathogens to such agents. For example, the response of Ralstonia solanacearum, the tomato wilt pathogen, to antimicrobial metabolites of Pseudomonas fluorescens is unknown. Thus, we assessed the effects of P. fluorescens SN15-2 fermentation broth on R. solanacearum by transmission electron microscopy and transcriptome technology. RNA sequencing identified 109 and 155 genes that are significantly upregulated and downregulated, respectively, in response to P. fluorescens metabolites, many of which are associated with the cell membrane and cell wall, and with nucleotide acid metabolism, iron absorption, and response to oxidative stress. This study highlights the effectiveness of P. fluorescens metabolites against the tomato wilt pathogen and helps clarify the underlying molecular mechanisms.

Investigating genetic characteristics of hepatitis B virus-associated and -non-associated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a primary liver malignancy that mainly occurs in patients with chronic liver disease and cirrhosis. Risk factors for HCC include hepatitis B virus (HBV) infection. However, the specific role of HBV infection in HCC development is not yet completely understood. In order to reveal the effects of HBV on HCC, we compare the genes of HCC patients infected with HBV with those who are not infected. METHODS: We encoded the genes of these two types of HCC in databases using enrichment scores of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway terms. A random forest algorithm was employed in order to distinguish these two types in the classifier, and a series of feature selection approaches was used in order to select their optimal features. Novel HBV-associated and -non-associated HCC genes were predicted, respectively, based on their optimal features in the classifier. A shortest-path algorithm was also employed in order to find all of the shortest-paths genes connecting the known related genes. RESULTS: A total of 54 different features between HBV-associated and -non-associated HCC genes were identified. In total, 1236 and 881 novel related genes were predicted for HBV-associated and -non-associated HCC, respectively. By integrating the predicted genes and shortest path genes in their gene interaction network, we identified 679 common genes involved in the two types of HCC. CONCLUSION: We identified the significantly different genetic features between two types of HCC. We also predicted related genes for the two types based on their specific features. Finally, we determined the common genes and features that were involved in both of these two types of HCC.

Simultaneous expression of displayed and secreted antibodies for antibody screen.

The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS) analysis, as well as in conditioned medium in a secreted version for function analysis.

[Construction of full-length human bladder cancer-specific antibody libraries based on mammalian display technology].

OBJECTIVE: To construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells. METHODS: The total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies. RESULTS: The libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11). CONCLUSION: We have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor.

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

[Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries].

OBJECTIVE: To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries. METHODS: Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry. RESULTS: The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10). CONCLUSION: The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.

[Preparation of human anti-HBs from volunteers with hepatitis B vaccine boost vaccination by modified B-cell immortalization technique].

OBJECTIVE: To establish immortalized B lymphoblast cell lines (B-LCLs) from healthy anti-HBs antibody (anti-HBs)-positive volunteers and screen for human anti-HBs and the antibody-secreting cells. METHODS: The peripheral blood mononuclear cells (PBMC) isolated from 3 healthy volunteers positive for anti-HBs with hepatitis B vaccine boost vaccination were infected with Epstein-Barr virus (EBV) and incubated in the presence of CpG DNA motifs and cyclosporin A (CyA). The anti-HBs in the culture supernatant of the immortalized B-cells was quantified by Architect anti-HBs assay with chemiluminescent microparticle technique. Immunocytochemistry was performed to identify the differentiation of the cell clones expressing anti-HBs. RESULTS: Immortalized B-cell culture was successfully established from the cell clones secreting anti-HBs with EBV infection and CpG DNA stimulation. The titer of anti-HBs in the culture supernatant was at its peak at 3 weeks of cell culture and then decreased gradually. At 3 months of cell culture, the cells still retained the capacity of anti-HBs production as verified by the results of immunocytochemistry for CD20 and CD138. CONCLUSION: Immortalized B-cell culture secreting anti-HBs from volunteers receiving boost hepatitis B vaccination has been successfully established by modified EBV immortalization technique.