Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood.

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.

[Quality evaluation and grading standard discussion on Cinnamomi Ramulus].

This study explored the relationship between the appearance traits and internal components of Cinnamomi Ramulus pieces, so as to provide a reference for the quality evaluation and the formulation of grade standards. This study determined the appearance traits and index component contents of 41 batches of Cinnamomi Ramulus pieces in the core producing areas of Guangxi and Guangdongand established the HPLC characteristic map method. The weight of the pieces, the narrowest diameter, and the widest diameter of the tr ansverse section were used as the indices of appearance traits. The content of index components(cinnamic acid and cinnamalde hyde)was determined by the established content determination method. The chromatographic characteristics were determinedon a Waters XBridge C_(18)(4. 6 mm×250 mm, 5 µm) column with a mobile phase consisting of 0. 1% phosphoric acidacetonitrile and gradient elution at the flow rate of 1 mL ·min~(-1). The column temperature was 30 ℃, and the detection wavelength was 254 nm. Cluster analysis, principal component analysis, and other stoichiometric methods were used to analyze the correlation between theap pearance traits and the index/characteristic components of Cinnamomi Ramulus pieces and compare the qu ality differences of the piecesfrom different batches and plac es. The results showed that the larger weight, the narrowest diameter, andthe widest diameter of the tra nsverse section indicated lowercontent of main indexes/characteristic components, and there was a synergistic decreasing trend amongd ifferent components. The overall quality of Cinnamomi Ramulus pieces in Guangdong Province and Guangxi Province was similar, but there were still differences between different origins and different batches of the same origin. It is scientific and feasible to evaluatethe quality of Cinnamomi Ramulus pieces and establish grading standards based on the appearance traits and index/character istic components. The research provides a more scientific and comprehensive basis for the quality control evaluation and standardformulation of Cinnamomi Ramulus.

Systematic Quality Evaluation of Epimedium wushanense T. S. Ying Based on Two Quality Control Standards: Total Flavonoid Glycosides and Epimedin C.

Two quality control standards, total flavonoid glycosides of Epimedii Folium and epimedin C of Epimedii Wushanensis Folium, were used to systematically evaluate the quality of Epimedium wushanense T. S. Ying, so as to provide reference for its germplasm screening and resource utilization. Seven representative populations of E. wushanense covering its main distribution areas were uniformly sampled during the flowering period. There were significant quality differences among the populations of E. wushanense. According to the quality standard of total flavonoid glycosides, all populations were superior to the quality standard of the Chinese Pharmacopoeia for Epimedii Folium, with more than 1.5 % total flavonoid glycosides. The variation ranges of epimedin A, epimedin B, epimedin C, icariin and total flavonoid glycosides were 0.40-0.76 %, 0.51-0.83 %, 1.70-9.31 %, 0.40-1.23 % and 3.05-10.61 %, respectively. According to the quality standard of epimedin C, all populations were better than the quality standard of the Chinese Pharmacopoeia for Epimedii Wushanensis Folium, with more than 1.0 % epimedin C. The variation range of epimedin C was 2.22-10.06 %. When comparing the results of the two methods, a trend of slightly lower mean values was found for total flavonoid glycosides, except for the HBXW population. The quality of E. wushanense was superior to both the quality standard of Epimedii Folium and Epimedii Wushanense Folium in the Chinese Pharmacopoeia. Epimedin C was the most abundant component. Among the investigated populations, HBXW and HBGK exhibited the highest quality, and may provide excellent genetic resources for standardized cultivation. In addition, the habitat of these populations can also serve a reference for cultivation conditions.

Muscular injury was identified the risk factor of post-operative stenosis after large area but non-circumferential esophageal endoscopic submucosal dissection.

BACKGROUND: The incidence of post-operative stenosis after esophageal Endoscopic Submucosal Dissection (ESD) ranks high. This study aimed to investigate the association between the degree of muscular injury and the incidence of post-operative stenosis. RESEARCH DESIGN: This was a retrospective study of 133 patients with superficial esophageal lesions treated by non-circumferential ESD enrolled between January 2016 and May 2021 at two endoscopy centers. Demographic and clinical parameters were analyzed. A novel muscular injury classification system was proposed. Stenosis risk factors were identified using multivariate logistic regression. The association between the different degrees of muscular injury and the incidence of post-operative stenosis was investigated further by propensity score matching (PSM). RESULTS: There were 133 cases evaluated in this study, 33 of which developed stenosis. Multivariate analysis suggested lesions located in the upper 1/3 of the esophagus, resections >5/6 of the circumference, and muscular injury (Grade 3 or 4 according to our proposed classification) were risk factors for stenosis. Correlation analysis suggested a positive association between the degree of muscular injury and the incidence of post-operative stenosis (r = 0.408, p < .05). For PSM, 29 stenosis cases were matched and univariate analysis further corroborated that muscular injuries of grade 3 (OR = 6.429, 95%CI = 1.318-31.367, p = .021) or 4 (OR = 7, 95%CI = 1.068-45.901, p = .043) were risk factors for stenosis. CONCLUSION: Grade 3-4 muscular injury was identified as a risk factor of post-operative stenosis.

USP22 deficiency in melanoma mediates resistance to T cells through IFNγ-JAK1-STAT1 signal axis.

Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated 9 (Cas9)-mediated loss-of-function screens are powerful tools for identifying genes responsible for diverse phenotypes. Here, we perturbed genes in melanoma cells to screen for genes involved in tumor escape from T cell-mediated killing. Multiple interferon gamma (IFNγ) signaling-related genes were enriched in melanoma cells resistant to T cell killing. In addition, deletion of the deubiquitinating protease ubiquitin specific peptidase 22 (USP22) in mouse melanoma (B16-OVA) cells decreased the efficacy of T cell-mediated killing, both in vitro and in vivo, while overexpression enhanced tumor-cell sensitivity to T (OT-I) cell-mediated killing. USP22 deficiency in both mouse and human melanoma cells showed impaired sensitivity to interferon pathway and USP22 was positively correlated with key molecules of interferon pathway in clinical melanoma samples. Mechanistically, USP22 may directly interact with signal transducer and activator of transcription 1 (STAT1), deubiquitinate it, and improve its stability in both human and mouse melanoma cells. Our findings identified a previously unknown function of USP22 and linked the loss of genes in tumor cells that are essential for escaping the effector function of CD8+ T cells during immunotherapy.

Chemical Constituents of the Marine Fungus Penicillium sp. MCCC 3A00228.

One new (d-arabinitol-anofinicate, 1) and fourteen known (2-15) compounds were isolated from the marine Penicillium sp. MCCC 3A00228. The structure of the new compound was established mainly by extensive spectroscopic analyses. Compound 1 exhibited weak transcriptional effect on Nur77. While compound 13 showed moderate in vitro anti-proliferative effect against QGY7701, H1299, and HCT116 tumor cells with IC50 values of 21.2 µM, 18.2 µM, and 17.6 µM, respectively.

Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays.

BACKGROUND: Cymbidium faberi is one of the oldest cultivars of oriental orchids, with an elegant flower fragrance. In order to investigate the molecular mechanism and the functions of related proteins in the methyl jasmonate (MeJA) signaling pathway, one of the main components of flower fragrance in C. faberi, yeast one- and two-hybrid three-frame cDNA libraries were constructed. RESULTS: In this study, a modified cDNA library used for yeast one- and two-hybrid screening was successfully constructed, with a recombinant efficiency of 95%. The lengths of inserted fragments ranged from 750~3000 bp, and the library capacity reached 6 × 109 CFU/ µg of cDNA insert, which was suitable for the requirements of subsequent screening. Finally, a homologous protein related with pathogenesis was screened out by the bait vector of CfbHLH36, which may participate in the MeJA signaling pathway. CONCLUSION: The yeast one- and two-hybrid library of C. faberi provides large amounts of useful information for the functional genomics research in C. faberi, and this method could also be applied to other plants to screen DNA-protein and protein-protein interactions.

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from L-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from L-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on L-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. KEY POINTS: • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine.

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle.

Bioplastics produced from microbial source are promising green alternatives to traditional petrochemical-derived plastics. Nonnatural straight-chain amino acids, especially 5-aminovalerate, 6-aminocaproate and 7-aminoheptanoate are potential monomers for the synthesis of polymeric bioplastics as their primary amine and carboxylic acid are ideal functional groups for polymerization. Previous pathways for 5-aminovalerate and 6-aminocaproate biosynthesis in microorganisms are derived from L-lysine catabolism and the citric acid cycle, respectively. Here, we show the construction of an artificial iterative carbon-chain-extension cycle in Escherichia coli for simultaneous production of a series of nonnatural amino acids with varying chain length. Overexpression of L-lysine α-oxidase in E. coli yields 2-keto-6-aminocaproate (2K6AC) as a non-native substrate for the artificial iterative carbon-chain-extension cycle. The chain-extended α-ketoacid products are decarboxylated and oxidized by an α-ketoacid decarboxylase and an aldehyde dehydrogenase, respectively, to yield their corresponding nonnatural straight-chain amino acids. The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of 5-aminovalerate, 46.96 mg/L of 6-aminocaproate and 4.78 mg/L of 7-aminoheptanoate after 8 h of enzyme catalysis starting from 2K6AC as the substrate. Furthermore, simultaneous production of 2.15 g/L of 5-aminovalerate, 24.12 mg/L of 6-aminocaproate and 4.74 mg/L of 7-aminoheptanoate was achieved in engineered E. coli. This work illustrates a promising metabolic-engineering strategy to access other medium-chain organic acids with -NH2, -SCH3, -SOCH3, -SH, -COOH, -COH, or -OH functional groups through carbon-chain-elongation chemistry.

Development of Plastid Genomic Resources for Discrimination and Classification of Epimedium wushanense (Berberidaceae).

Epimedium wushanense (Berberidaceae) is recorded as the source plant of Epimedii Wushanensis Folium in the Chinese Pharmacopoeia. However, controversies exist on the classification of E. wushanense and its closely related species, namely, E. pseudowushanense, E. chlorandrum, E. mikinorii, E. ilicifolium, and E. borealiguizhouense. These species are often confused with one another because of their highly similar morphological characteristics. This confusion leads to misuse in the medicinal market threatening efficiency and safety. Here, we studied the plastid genomes of these Epimedium species. Results show that the plastid genomes of E. wushanense and its relative species are typical circular tetramerous structure, with lengths of 156,855-158,251 bp. A total of 112 genes were identified from the Epimedium plastid genomes, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A loss of rpl32 gene in E. chlorandrum was found for the first time in this study. The phylogenetic trees constructed indicated that E. wushanense can be distinguished from its closely related species. E. wushanense shows a closer relationship to species in ser. Dolichocerae. In conclusion, the use of plastid genomes contributes useful genetic information for identifying medicinally important species E. wushanense and provides new evidence for understanding phylogenetic relationships within the Epimedium genus.

[Pharmacognostical study on four origin plants of folk medicine Sikuaiwa].

In order to develop characteristic folk medicine resources in Jiangxi, a pharmacognostical study was systematically performed for four different origin plants of Sikuaiwa, the result of study provides the microscopic features of powder and tissue of the crude drug. The research provided reference for the identification of Sikuaiwa, as well as a theoretical basis for the further development and the formulation of quality standards.

Genetic Diversity Analysis and Core Germplasm Construction of Rubus chingii Hu.

Rubus chingii Hu is the only species that is used for both edible and medicinal purposes among the 194 species of the genus Rubus in China. It is well known for its sweet and sour fresh fruits that are rich in vitamins and for its dried immature fruits that are used to treat kidney-related ailments. This study aims to evaluate genetic diversity and population structure and build a core germplasm repository of 132 R. chingii accessions from the provinces of Jiangxi and Fujian, using Hyper-seq-derived single-nucleotide polymorphism (SNP) markers. This is the first genetic study of R. chingii based on SNP molecular markers, and a total of 1,303,850 SNPs and 433,159 insertions/deletions (InDels) were identified. Low values for observed heterozygosity, nucleotide diversity (Pi) and fixation indexes (Fis) indicated low genetic diversity within populations, and an analysis of molecular variance (AMOVA) showed that 37.4% and 62.6% of the variations were found between populations and within samples, respectively. Four main clusters were identified by means of neighbor-joining (NJ) trees, the ADMIXTURE program and principal component analysis (PCA). Based on the genetic diversity, we finally constructed 38 representative core collections, representing 50% of the total core germplasm samples and 95.3% of the genotypes. In summary, the results of our study can provide valuable information on the genetic structure of R. chingii germplasm resources, which is helpful for further explorations of potential high-quality genes and for formulating future breeding and conservation strategies.

Metabolomic and transcriptomic analysis of the flavonoid biosynthesis pathway in Epimedium sagittatum (Sieb. et Zucc.) Maxim. from distinct locations.

Epimedium sagittatum (Sieb. et Zucc.) Maxim. (ESM) which accumulates several principal flavonoid compounds including epimedin A, B, C and icariin, is extensively utilized in traditional herbs for sexual dysfunction, osteoporosis etc. In China, ESM has a wealth of wild plant resources and characterized by significant variability in medicinal compounds accumulation. Understanding the diversity of ESMs can lead to better utilization of these plant resources. In this study, we integrated the metabolomic and transcriptomic analysis of three ESMs that originated in Anhui, Hubei and Jiangxi in China. Results showed that the flavonoid biosynthesis as well as the related gene expression in these ESMs revealed substantial differences. For example, the epimedin A, B, C and icariin as well as some related gene expression in ESMs from Anhui are significantly lower than those of in others. These results suggested that the ESMs from wild population without quality checkout may not be suitable for directly use as the materials for preparation of Chinese medicine and ESMs with different accumulation of metabolites could be used for distinct applications.

Variation of epimedins A - C and icariin in ten representative populations of Epimedium brevicornu Maxim., and implications for utilization.

The concentration variations of main flavonoids, epimedins A-C and icariin, among ten representative populations of Epimedium brevicornu Maxim. were assessed by HPLC. The populations were collected during the flowering stage and included 419 individual samples. Remarkable variations within and among populations were detected. SXXA Population (see Fig. 1) was an outlier due to its significant low concentrations (<1.00-4.46 mg/g). But even without SXXA, significant concentration differences among populations were still observed in epimedin A (2.31-8.42 mg/g), epimedin B (6.67-55.7 mg/g), epimedin C (5.39-23.0 mg/g), icariin (8.50-39.9 mg/g), and their total (29.1-123 mg/g). All populations except SXXA showed much higher concentrations than the recommended standards (i.e. 5 mg/g for icariin and 13 mg/g for the total). A high-concentration-population structure, estimated both by principal component analysis (PCA) and unweighted pair group method with averaging (UPGMA) cluster analysis, based on Euclidean distances, was observed. Both methods allowed separation of the populations in four groups defined by the concentrations of four main flavonoids. The populations (SXLC and SXQS) located in north of Yellow River were clustered together and characterized by highest concentrations of epimedin B, icariin, and their total. Considering of the high concentrations of main flavonoids and abundant resources, E. brevicornu could be exploited as a good medical resource for Herba Epimedii and would offer a tremendous potential for commercial development, but SXXA population should be paid special attention, and further study is needed.

Prognostic value and chemotherapy response prediction of a proliferation essential gene signature in colon cancer.

BACKGROUND: Colon cancer is a common malignant tumor in the digestive tract. Exploring new treatment targets is of great significance for improving the survival rate of colon cancer patients. The present study mainly analyzes the impact of proliferation essential genes (PLEGs) on the prognosis and chemotherapy response of colon cancer patients, as well as identifying the expression and cellular functions of important PLEG. METHODS: The DepMap database was utilized for identification of PLEG in colon cancer cells. Through DEGs screening, WGCNA, univariate cox regression survival analysis, and LASSO, a PLEG signature (PLEGs) model was constructed. The impact of PLEGs on the prognosis of colon cancer patients and their response to chemotherapy was further analyzed. Finally, we conducted a random forest analysis and implemented functional experiments to investigate the prominent PLEG that is linked to the development of colon cancer. RESULTS: Based on the expression and prognosis of PLEG, we constructed a PLEGs prognosis model which can effectively predict the prognosis of colon cancer patients and their response to chemotherapy treatment. Random forest analysis showed that UBA1 is a key PLEG in the progression of colon cancer. Immunohistochemistry results revealed that UBA1 protein is significantly upregulated in colon cancer tissues. Cell functional experiments demonstrated that knocking down UBA1 can inhibit the proliferation, invasion, and migration abilities of colon cancer cells. CONCLUSION: PLEGs have the potential to serve as predictive biomarkers for prognosis and chemotherapy response in colon cancer patients. Among the PLEG, UBA1 plays a prominent role in promoting the malignant progression of colon cancer cells.

Development and Validation of a Risk Scoring Tool for Bronchopulmonary Dysplasia in Preterm Infants Based on a Systematic Review and Meta-Analysis.

Background: Bronchopulmonary dysplasia (BPD) is the most common serious pulmonary morbidity in preterm infants with high disability and mortality rates. Early identification and treatment of BPD is critical. Objective: This study aimed to develop and validate a risk scoring tool for early identification of preterm infants that are at high-risk for developing BPD. Methods: The derivation cohort was derived from a systematic review and meta-analysis of risk factors for BPD. The statistically significant risk factors with their corresponding odds ratios were utilized to construct a logistic regression risk prediction model. By scoring the weights of each risk factor, a risk scoring tool was established and the risk stratification was divided. External verification was carried out by a validation cohort from China. Results: Approximately 83,034 preterm infants with gestational age < 32 weeks and/or birth weight < 1500 g were screened in this meta-analysis, and the cumulative incidence of BPD was about 30.37%. The nine predictors of this model were Chorioamnionitis, Gestational age, Birth weight, Sex, Small for gestational age, 5 min Apgar score, Delivery room intubation, and Surfactant and Respiratory distress syndrome. Based on the weight of each risk factor, we translated it into a simple clinical scoring tool with a total score ranging from 0 to 64. External validation showed that the tool had good discrimination, the area under the curve was 0.907, and that the Hosmer-Lemeshow test showed a good fit (p = 0.3572). In addition, the results of the calibration curve and decision curve analysis suggested that the tool showed significant conformity and net benefit. When the optimal cut-off value was 25.5, the sensitivity and specificity were 0.897 and 0.873, respectively. The resulting risk scoring tool classified the population of preterm infants into low-risk, low-intermediate, high-intermediate, and high-risk groups. This BPD risk scoring tool is suitable for preterm infants with gestational age < 32 weeks and/or birth weight < 1500 g. Conclusions: An effective risk prediction scoring tool based on a systematic review and meta-analysis was developed and validated. This simple tool may play an important role in establishing a screening strategy for BPD in preterm infants and potentially guide early intervention.

Injectable, Hierarchically Degraded Bioactive Scaffold for Bone Regeneration.

Bioactive materials play vital roles in the repair of critical bone defects. However, bone tissue engineering and regenerative medicine are still challenged by the need to repair bone defects evenly and completely. In this study, we functionally simulated the natural creeping substitution process of autologous bone repair by constructing an injectable, hierarchically degradable bioactive scaffold with a composite hydrogel, decalcified bone matrix (DBM) particles, and bone morphogenetic protein 2. This composite scaffold exhibited superior mechanical properties. The scaffold promoted cell proliferation and osteogenic differentiation through multiple signaling pathways. The hierarchical degradation rates of the crosslinked hydrogel and DBM particles accelerated tissue ingrowth and bone formation with a naturally woven bone-like structure in vivo. In the rat calvarial critical defect repair model, the composite scaffold provided even and complete repair of the entire defect area while also integrating the new and host bone effectively. Our results indicate that this injectable, hierarchically degradable bioactive scaffold promotes bone regeneration and provides a promising strategy for evenly and completely repairing the bone defects.