Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

LncRNA MEG3 inhibits renal fibrinoid necrosis of diabetic nephropathy via the MEG3/miR-21/ORAI1 axis.

INTRODUCTION: Diabetic nephropathy (DN) is one of the most common and lethal diabetic complications worldwide and is associated with a high risk of mortality. However, the exact mechanism behind its development is unknown. The mesangial cells (MCs) and non-coding RNAs are critical for DN, but it is unknown whether a MEG3/miR-21/ORAI1 regulatory axis exists in MCs. Hence, in this study, we aimed to understand whether the MEG3/miR-21/ORAI1 regulatory axis has a role in the pathophysiology of DN. RESULTS: We demonstrated that high-glucose stimuli downregulated MEG3 and ORAI1 expression while enhancing miR-21 expression. Exogenous miR-21 mimics inhibited ORAI1 expression, which was partially salvaged or reversed by MEG3 overexpression. Furthermore, RIP assay demonstrated that the beads labeled with AGO2 antibody could enrich more miR-21 and MEG3 than those labeled with control IgG antibody; both of them formed the RNA-induced silencing complex. Further, the biochemical indicators of db/db mice significantly improved, and renal fibrinoid necrosis was ameliorated using a miR-21 inhibitor. CONCLUSION: The MEG3/miR-21/ORAI1 axis regulates the manifestation of DN in diabetic mice and MCs, and the miR-21 inhibitor can be a potential therapeutic strategy to alleviate DN, once the presence of such an axis is found in humans.

Effects of hypoxia and reoxygenation on oxidative stress, histological structure, and apoptosis in a new hypoxia-tolerant variety of blunt snout bream (Megalobrama amblycephala).

A new hypoxia-tolerant variety of blunt snout bream was obtained by successive breeding of the wild population, which markedly improved hypoxia tolerance. In this study, the hypoxia-tolerant variety was exposed to hypoxia (2.0 mg O2·L-1) for 4, 7 days. The contents of blood biochemical indicators including the number of red blood cells (RBC), total cholesterol (T-CHO), total protein (TP), triglyceride (TG), glucose (GLU), and lactic acid (LD) increased significantly (P < 0.05) under hypoxia. The glycogen content in the liver and muscle decreased significantly (P < 0.05) and the LD content in the brain, muscle and liver increased significantly (P < 0.05) under hypoxia. The levels of oxidative stress-related indicators i.e., superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and total antioxidant capacity (T-AOC) also changed significantly (P < 0.05) in the heart, liver, and intestine of the new variety under hypoxia. Additionally, hypoxia has caused injuries to the heart, liver, and intestine, but it shows amazing repair ability during reoxygenation. The apoptotic cells and apoptosis rate in the heart, liver, and intestine increased under hypoxia. Under hypoxia, the expression of the B-cell lymphomas 2 (Bcl-2) gene in the heart, liver, and intestine was significantly (P < 0.05) down-regulated, while the expression of the BCL2-associated agonist of cell death (Bad) gene was significantly (P < 0.05) up-regulated. These results are of great significance for enriching the basic data of blunt snout bream new variety in response to hypoxia and promoting the healthy development of its culture industry.

Functional exploration of SNP mutations in HIF2αb gene correlated with hypoxia tolerance in blunt snout bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A203A752) and its homozygous diplotype II (A203A203A752A752) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOEcrit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48 h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48 h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.

Effects of hypoxia on the gill morphological structure, apoptosis and hypoxia-related gene expression in blunt snout bream (Megalobrama amblycephala).

The blunt snout bream (Megalobrama amblycephala) is a typical hypoxia-sensitive fish, and hypoxia stress leads to reduced vitality and yield during aquaculture. To explore the specific adaptation mechanism under hypoxia, the blunt snout bream was treated with hypoxia (DO = 2.0 ± 0.1 mg/L) for 24 h, followed by 3 h of recovery. Our results depicted that the gill filament structure of blunt snout bream changed after hypoxia. During hypoxia for 24 h, the gill filament structure was altered, including a more than 80% expansion of the lamellar respiratory surface area and a proportionate apoptosis decrease in interlamellar cell mass (ILCM) volume. Thus, the water-blood diffusion distance was shortened to less than 46%. During hypoxia for 24 h, the activity of ROS in gill tissue increased significantly (p < 0.05), while the mitochondrial membrane potential decreased significantly (p < 0.05). During hypoxia, mRNA expression level of anti-apoptotic gene Bcl-2 in the gills of blunt snout bream decreased significantly (p < 0.05), while the expression of pro-apoptotic gene Bax mRNA increased significantly (p < 0.05). Thus, the ratio of Bax/Bcl-2 mRNA increased in the gills of blunt snout bream to promote the activity of Caspase-3. Together, our results indicated hypoxia-induced apoptosis in the gills of blunt snout bream through the mitochondrial pathway. In addition, a decreased expression of Phd1 and an increased expression of Hif-1α in gills under hypoxia stress indicates that blunt snout bream may cope with hypoxia-induced apoptosis by enhancing the HIF pathway. These results provide new insights into fish's adaptation strategies and mechanisms of hypoxia.

[Latest Findings on NOD-Like Receptor Family Pyrin Domain Containing Protein 3 Inflammasome and Bone and Articular Diseases].

Inflammasomes are important components of the innate immune system. They are assembled by cytoplasmic pattern recognition receptors and play a critical role in the pathogenesis and progression of various inflammatory diseases through regulating the release and activation of inflammatory cytokines and inducing cell prytosis. NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome has been widely studied and has been shown to be closely associated with cardiovascular diseases and metabolic disorders. Bone and joint diseases, such as osteoarthritis and rheumatoid arthritis show high prevalence worldwide and can cause bone and cartilage damage, pain, and dysfunction, adversely affecting the patients' quality of life. The reported findings of some studies indicate that the pathogenesis of various bone and articular diseases is associated with NLRP3 inflammasome. Small molecule antagonists targeting NLRP3 inflammasome have shown considerable therapeutic potentials, but their clinical application still needs further exploration. Herein, we reviewed the composition and function of NLRP3 inflammasome and its association with bone and articular diseases.

[Research progress on traditional Chinese medicine compounds combined with external therapy in treatment of hyperplasia of mammary glands].

Hyperplasia of mammary glands is a benign breast disease with disordered breast structure. Nowadays, the incidence rate of breast hyperplasia in women is increasing year by year, and the etiology is related to the imbalance of estrogen and progesterone in the body. The symptoms include breast pain, breast nodules, or nipple discharge, which can develop into breast cancer in the context of psychological pressure. Therefore, it is timely and effectively necessary for people to treat the symptoms. At present, traditional Chinese medicine(TCM) often treats breast hyperplasia by oral drug, external application, acupuncture, moxibustion, and massage, while western medicine often uses hormone therapy or surgery. TCM can regulate hormone levels to treat breast hyperplasia. Acupuncture, moxibustion, and other methods can stimulate acupoints to reduce breast lumps. However, since TCM is easy to produce hepatorenal toxicity after long-term use and simple external treatment is slow to take effect, rapid and effective treatment is difficult to be achieved. Although western medicine can inhibit the disease, it is easy to produce toxic and side effects if taken for a long time. In addition, surgery can only remove the focus and the recurrence rate is high. Some studies have found that the combination of oral and external use of TCM compounds has a significant effect, with mild toxic and side effects, few adverse reactions, and a low recurrence rate. Based on the relevant literature in recent years, this article reviewed the combination of oral and external treatment of TCM in the treatment of hyperplasia of mammary glands, discussed the effectiveness, clinical evaluation indexes, and mechanism, and pointed out the existing shortcomings to explore a comprehensive therapy worthy of clinical application.

The transcriptomic responses of blunt snout bream (Megalobrama amblycephala) to acute hypoxia stress alone, and in combination with bortezomib.

BACKGROUND: Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia. A new blunt snout bream strain, "Pujiang No.2", was developed to overcome this shortcoming. As a proteasome inhibitor, bortezomib (PS-341) has been shown to affect the adaptation of cells to a hypoxic environment. In the present study, bortezomib was used to explore the hypoxia adaptation mechanism of "Pujiang No.2". We examined how acute hypoxia alone (hypoxia-treated, HN: 1.0 mg·L- 1), and in combination with bortezomib (hypoxia-bortezomib-treated, HB: Use 1 mg bortezomib for 1 kg fish), impacted the hepatic ultrastructure and transcriptome expression compared to control fish (normoxia-treated, NN). RESULTS: Hypoxia tolerance was significantly decreased in the bortezomib-treated group (LOEcrit, loss of equilibrium, 1.11 mg·L- 1 and 1.32 mg·L- 1) compared to the control group (LOEcrit, 0.73 mg·L- 1 and 0.85 mg·L- 1). The HB group had more severe liver injury than the HN group. Specifically, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the HB group (52.16 U/gprot, 32 U/gprot) were significantly (p < 0.01) higher than those in the HN group (32.85 U/gprot, 21. 68 U/gprot). In addition, more severe liver damage such as vacuoles, nuclear atrophy, and nuclear lysis were observed in the HB group. RNA-seq was performed on livers from the HN, HB and NN groups. KEGG pathway analysis disclosed that many DEGs (differently expressed genes) were enriched in the HIF-1, FOXO, MAPK, PI3K-Akt and AMPK signaling pathway and their downstream. CONCLUSION: We explored the adaptation mechanism of "Pujiang No.2" to hypoxia stress by using bortezomib, and combined with transcriptome analysis, accurately captured the genes related to hypoxia tolerance advantage.

Chromosome-level genome assembly of grass carp (Ctenopharyngodon idella) provides insights into its genome evolution.

BACKGROUND: The grass carp has great economic value and occupies an important evolutionary position. Genomic information regarding this species could help better understand its rapid growth rate as well as its unique body plan and environmental adaptation. RESULTS: We assembled the chromosome-level grass carp genome using the PacBio sequencing and chromosome structure capture technique. The final genome assembly has a total length of 893.2 Mb with a contig N50 of 19.3 Mb and a scaffold N50 of 35.7 Mb. About 99.85% of the assembled contigs were anchored into 24 chromosomes. Based on the prediction, this genome contained 30,342 protein-coding genes and 43.26% repetitive sequences. Furthermore, we determined that the large genome size can be attributed to the DNA-mediated transposable elements which accounted for 58.9% of the repetitive sequences in grass carp. We identified that the grass carp has only 24 pairs of chromosomes due to the fusion of two ancestral chromosomes. Enrichment analyses of significantly expanded and positively selected genes reflected evolutionary adaptation of grass carp to the feeding habits. We also detected the loss of conserved non-coding regulatory elements associated with the development of the immune system, nervous system, and digestive system, which may be critical for grass carp herbivorous traits. CONCLUSIONS: The high-quality reference genome reported here provides a valuable resource for the genetic improvement and molecular-guided breeding of the grass carp.

An SNP at the target site of cid-miR-nov-1043 in the TOLLIP 3' UTR decreases mortality rate in grass carp subjected to ENU-induced mutagenesis following grass carp reovirus infection.

N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3' UTR of Toll interacting protein gene (TOLLIP982T>C) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3' UTR, but TOLLIP982T>C mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3' UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor ß-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP982T>C mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP982T>C mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3' UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp.

Highly efficient blue quantum-dot light-emitting diodes based on a mixed composite of a carbazole donor and a triazine acceptor as the hole transport layer.

Solution-processed thermally activated delayed fluorescence (TADF) exciplexes were employed as the hole transport layer (HTL) of blue quantum dot (QD) light-emitting diodes (QLEDs) by blending polymer donors of poly(N-vinylcarbazole) (PVK) with small molecular acceptors of 2,4,6-tris(biphenyl-3-yl)-1,3,5-triazine (T2T). As a result, the PVK:T2T HTL can harvest holes and electrons leaking from the QD active layer to form exciplex excitons and then this harvested exciton energy can be effectively transferred to the adjacent QD emitters through the Förster resonance energy-transfer process. Furthermore, the TADF exciplexes can enhance the hole mobility of the HTL due to the charge transfer process from the PVK donor to the T2T acceptor under an external electric field. The maximum current efficiency (CE) and external quantum efficiency (EQE) of the fabricated blue ZnCdS/ZnS core/shell QLEDs increase from 4.14 cd A-1 and 7.33% for the PVK HTL to 7.73 cd A-1 and 13.66% for the PVK:(5 wt%)T2T HTL, respectively. Our results demonstrate that the TADF exciplex HTL would be a facile strategy to design high-performance blue QLEDs.

Gill remodeling increases the respiratory surface area of grass carp (Ctenopharyngodon idella) under hypoxic stress.

Seasonal changes, diurnal variations, and eutrophication result in periodic hypoxia in fish habitats, thus affecting the success of commercial aquaculture. In this study, the grass carp (Ctenopharyngodon idella) presented moderate hypoxia tolerance; they showed a medium critical oxygen tension during the loss of equilibrium. In response to 7 d of hypoxic exposure, the erythrocyte count and hemoglobin (Hb) concentration significantly increased (p < 0.01). To cope with the hypoxic environment, the grass carp underwent gill remodeling marked by reduction in the interlamellar cell mass (ILCM) and an increase in respiratory surface area. The gill remodeling under hypoxia was enabled by apoptosis induction. Although apoptotic signals were not found on ILCM cells, transferase dUTP nick end labeling (TUNEL) assay results indicated that after 1 d of hypoxic exposure, the number of TUNEL-positive cells per lamella increased until 4 d and then began to decrease. Consistent with the results of the TUNEL assay, the mRNA expression of apoptosis-related genes, caspase-3, Bax, and Bcl-2, increased at 1, 4, and 7 d of the hypoxia treatment. In addition, gill remodeling significantly (p < 0.01) decreased the concentration of sodium and chloride ions in the fish serum. These findings provide evidence that grass carps increase their respiratory surface area through gill remodeling by apoptosis in the gill filaments to acclimate to a hypoxic environment. This study expands our understanding of the morphological and physiological changes in grass carp in response to a hypoxic environment; therefore, it could be useful for maintaining grass carp production.

Bacterial dynamics and functions driven by bulking agents to enhance organic degradation in food waste in-situ rapid biological reduction (IRBR).

This study investigated the effects of different bulking agents (i.e., sawdust, wheat straw, rice straw, and corncob) on bacterial structure and functions for organic degradation during food waste in-situ rapid biological reduction (IRBR) inoculated with microbial agent. Results showed that the highest organic degradation (409.5 g/kg total solid) and volatile solids removal efficiency (41.0%) were achieved when wheat straw was used, largely because the degradation of readily degradable substrates and cellulose was promoted by this bulking agent. Compared with other three bulking agents, the utilization of wheat straw was conducive to construct a more suitable environmental condition (moisture content of 18.0-28.2%, pH of 4.91-5.87) for organic degradation during IRBR process, by virtue of its excellent structural and physiochemical properties. Microbial community analysis suggested that the high-moisture environment in rice straw treatment promoted the growth of Staphylococcus and inhibited the activity of the inoculum. By contrast, lowest bacterial richness was observed in corncob treatment due to the faster water loss. Compared with these two bulking agents, sawdust and wheat straw treatment led to a more stable bacterial community structure, and the inoculated Bacillus gradually became the dominant genus (36.6-57.8%) in wheat straw treatment. Predicted metagenomics analysis showed that wheat straw treatment exhibited the highest carbohydrate metabolism activity which improved the pyruvate, amino sugar and nucleotide sugar metabolism, and thereby promoted the organic degradation and humic substrate production. These results indicated that wheat straw was a more desirable bulking agent, and revealed the potential microbial organics degradation mechanism in IRBR process.

Effects of hypoxia and reoxygenation on gill remodeling, apoptosis, and oxidative stress in hypoxia-tolerant new variety blunt snout bream (Megalobrama amblycephala).

Blunt snout bream plays an important role in freshwater aquaculture in China, but the development of its culture industry has been restricted by increasing hypoxia problem. Through the breeding of wild blunt snout bream populations (F0), a hypoxia-tolerant new variety (F6) was obtained. In this study, the new variety was stressed under low oxygen concentration (2.0 mg·L-1) for 4 and 7 days, the morphological structure of the gill tissue showed a striking change, the interlamellar cell mass (ILCM) volume reduced significantly (P < 0.05), and the lamellar respiratory surface area enlarged significantly (P < 0.05), compared to normoxic controls. After 7 days of oxygen recovery, gill remodeling was completely reversed. Additionally, the TUNEL-positive apoptotic fluorescence signals increased in the gills exposed to hypoxia up to 4 and 7 days; the apoptosis rate also increased significantly (P < 0.05). Under 4 and 7 days of hypoxia stress, the expression of anti-apoptotic gene Bcl-2 in the gills downregulated significantly (P < 0.05), with the significantly (P < 0.05) upregulated expression of pro-apoptotic gene Bad. Furthermore, under hypoxia stress, the activity or content of oxidative stress-related enzymes (superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and glutathione (GSH)) in gill tissue increased to varying degrees compared to normoxic controls. These results offer a new perspective into the cellular and molecular mechanism of hypoxia-induced gill remodeling in blunt snout bream and a theoretical basis for its hypoxia adaptation mechanism.

[Porphyromonas gingivalis HmuY and Its Possible Effect on the Pathogenesis of Periodontitis].

Periodontitis, one of the most common inflammatory oral diseases in human beings, threatens the health of teeth and mouth and is closely associated with the development of many systemic diseases. Existing research about the pathogenesis of periodontitis mainly focuses on the oral microbial homeostasis and its complex interaction with the immune system. Among all the oral microorganisms, Porphyromonas gingivalis ( P. gingivalis) is considered to be the main pathogen causing chronic periodontitis. Recent studies have shown that P. gingivalis poesseses HmuY, a special heme binding protein, which binds with heme to provide essential nutrition for P. gingivalis and activates the host immune system. Therefore, HmuY plays an important role in the growth, proliferation, invasion, and pathogenesis of P. gingivalis and is a potential virulence factor of the bacteria. Existing studies on HmuY are limited to the host immune response that HmuY triggers, and there are still no conclusive findings on whether HmuY participates in the pathogenesis of periodontitis through other ways, such as influencing periodontal bone metabolism. Herein, we reviewed the latest research findings on the biological characteristics and physiological functions of HmuY and its relationship with chronic periodontitis, so as to provide new ideas for in-depth research and further explorations into the pathogenesis of chronic periodontitis.

A novel prognostic index of hepatocellular carcinoma based on immunogenomic landscape analysis.

Changes in immune responses to hepatocellular carcinoma (HCC) are closely related to the occurrence, development, and prognosis of this disease. Exploring the role of immune-related genes (IRGs) in HCC would provide insights into the mechanisms regulating this disease. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) provide a platform for such research, owing to a large number of HCC samples available for comprehensive and systematic immunogenomics analyses. We analyzed the IRGs expression profile and clinical information of patients with HCC based on the TCGA and ICGC database. Potential molecular mechanisms and properties of the screened IRGs were analyzed across multiple databases. And we analyzed the correlation between IRGs, single-nucleotide polymorphisms, and copy number variation. A novel prognostic index, based on IRGs, was developed using the LASSO Cox regression algorithm, followed by univariate and multivariate Cox regression analyses to analyze the prognostic index. Information in the ICGC database was used to verify the reliability of the prognostic index. A total of 54 differentially expressed IRGs were found to be significantly associated with HCC prognosis, and there is a significant correlation between their expression level and copy number variation. Functional enrichment analyses indicated that the genes play active roles in tumor and immune-related signaling pathways. In addition, five potential biomarkers namely IRG, MAPK3, HSP90AA1, HSP90AB1, HSPA4, and CDK4, were identified. Finally, a novel prognostic index, based on IRGs (PSMD14, FABP6, ISG20L2, HGF, BIRC5, IL17D, and STC2), was found useful as an independent prognostic factor, not only for prognosis but also to reflect levels of infiltration in a variety of immune cells. Our team conducted a genomics study of IRGs in HCC and screened several clinically significant IRGs, and our model provides an effective approach for stratification and characterization of patients using IRG-based immunolabeling tools to monitor the prognosis of HCC.

Selection of functional EPHB2 genotypes from ENU mutated grass carp treated with GCRV.

BACKGROUND: N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. RESULTS: This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal's responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859G > A) and 5968 (5968G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. CONCLUSION: Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.

Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris.

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 µg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300 was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes.

High-level production of d-pantothenic acid from glucose by fed-batch cultivation of Escherichia coli.

d-Pantothenic acid (D-PA) is an essential vitamin widely used in food, feed, chemical, and pharmaceutical industries. An Escherichia coli platform was developed for the high-level production of D-PA from glucose through fed-batch cultivation. Initially, the effects of different glucose feeding strategies D-PA synthesis were studied. It was found that D-PA production in glucose control (5 g/L) fed-batch culture reached 24.3 g/L, which was 4.09 times that in the batch culture. Next, the effect of auxotrophic amino acid (isoleucine)-limited feeding on D-PA production was investigated. The results revealed that isoleucine feeding decreased the accumulation of by-product acetic acid and promoted D-PA production significantly. Furthermore, an isoleucine feeding embedded multistage glucose supply strategy was established, and a maximum titer of 39.1 g/L was achieved. To the best of our knowledge, this levels are the highest reported so far in engineered E. coli for the D-PA production. The developed fed-batch feeding strategy may be useful for the industrial D-PA production by E. coli.

[Research Progress in Mechanotransduction Process of Mechanical-Stress-Induced Autophagy].

As a self-protective mechanism for cells to obtain energy by degrading their own structures or substances, autophagy widely occurs in basic physiological process of all kinds of eukaryotic cells. In recent years, studies have shown that autophagy can be induced through a variety of mechanical transduction pathways when various tissues and cells are exposed to different types of mechanical stress, and cells and tissues involved can thus regulate cell metabolic functions and participate in the pathological process of a variety of diseases. The stress receptors on the cell membrane and the multiple signaling pathways and cytoskeletons have been shown to play an important role in this process. At present, due to the difficulties in the establishment of the stress loading model and the limitations in the research methods concerned, the specific mechanical transduction mechanisms of autophagy induced by mechanical stress is not clear. Therefore, more reliable in vitro and in vivo models and more advanced research methodology are needed to investigate the mechanical transduction process of autophagy induced by mechanical stress, and to promote ultimately progress in the understanding of autophagy-related diseases and their treatments. This article reviewed the regulatory role of mechanical stress on autophagy in physiological and disease processes and the signal transduction process related to autophagy induced by mechanical stress.