Results Of Assay Research Articles

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis

Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. CEPI.

Antibacterial Metabolites Produced by Limonium lopadusanum, an Endemic Plant of Lampedusa Island.

Lampedusa, the largest island of the Pelagie archipelago, Sicily, Italy, has proven to be a rich source of plants and shrubs used in folk medicine. These plants, often native to the island, have been very poorly investigated for their phytochemical composition and biological potential to be translated into pharmacological applications. To start achieving this purpose, a specimen of Limonium lopadusanum, a plant native to Lampedusa, was investigated for the first time. This manuscript reports the results of a preliminary biological assay, focused on antimicrobial activity, carried out using the plant organic extracts, and the isolation and chemical and biological characterization of the secondary metabolites obtained. Thus 3-hydroxy-4-methoxybenzoic acid methyl ester (syn: methyl isovanillate, (1), methyl syringate (2), pinoresinol (3), erythrinassinate C (4) and tyrosol palmitate (5) were isolated. Their antimicrobial activity was tested on several strains and compound 4 showed promising antibacterial activity against Enterococcus faecalis. Thus, this metabolite has antibiotic potential against the drug-resistant opportunistic pathogen E. faecalis.

Comprehensive genomic profiling of squamous cell carcinoma of unknown primary presenting with liver metastases.

755 Background: Carcinomas of unknown primary (CUPs) are comprised of metastatic cancers for which a primary tumor of origin cannot be identified despite a full diagnostic workup. They frequently present with liver metastases, which portend a poor prognosis. Strategies employing comprehensive genomic profiling (CGP) to identify targeted treatments for CUPs have shown potential. Despite this, the molecular landscape of squamous cell carcinomas of unknown primary (SCCUPs) remains poorly defined. This study describes the results of CGP testing in patients with SCCUPs identified in the Foundation Medicine, Inc. database, with a focus on those with liver involvement. Methods: Cases of SCCUPs with FoundationOne CDx (F1CDx) assay results were identified based on a reported diagnosis of CUP and the presence of SCC histology. Cases then underwent central pathologist review and were excluded if a known primary site could be determined based on histology and disease location. Cases with liver involvement were selected based on the site of tissue biopsy. Genomic analyses of identified cases were assessed using the FoundationCORE database. Results: Sixty-nine cases of SCCUP presenting with liver involvement were identified. Alterations were observed in 102 of 324 (31.5%) genes evaluated by the F1CDx assay in at least 1 patient. The most frequently altered genes were TP53 (66.7%) , CDKN2A (33.3%) , CDKN2B (23.2%) , KRAS (21.7%; 0% G12C) , PIK3CA (18.8%), NFE2L2 (11.6%), SOX2 (11.6%), M2M2 (10.1%), and MTAP (10.1%). No patients had microsatellite instability-high disease, only 6 (8.7%) patients had a tumor mutational burden (TMB) ≥ 10, and 11 (15.9%) patients had evidence of HPV infection. Among 33 patients with available PDL-1 testing results, 7 (21.2%) had high-positive expression. An additional 374 cases of SCCUP were identified with involvement outside of the liver. Compared to these cases, patients presenting with liver involvement had less genomic alterations per tumor (5.10 vs 6.71, p=0.005), and were less likely to have TMB ≥ 10 (8.7% vs 38.0%; odds ratio [OR] 0.16, p<0.001). KRAS mutations were observed in 21.7% of those presenting with liver metastases compared to 8.3% of other SCCUP patients (OR 3.07, p=0.07). Conclusions: SCCUPs presenting with liver metastases had reduced numbers of genomic alterations and decreased TMB compared to other SCCUPs, suggesting these tumors may be less likely to respond to immunotherapy. Despite this, 36.2% of these patients had at least one alteration that could potentially guide targeted treatment decisions including entry into biomarker-driven clinical trials. Interestingly, there was a trend toward increased KRAS alterations in those with liver metastases. To our knowledge, this is the first report that describes the genomic landscape of SCCUPs presenting with liver metastases.

Six bufadienolides derivatives are the main active substance against human colorectal cancer HCT116 cells of Huachansu injection

BackgroundHuachansu injection (HCS) is an aqueous extract preparation of Traditional Chinese Medicine (TCM) toad skin (Bufo melanosticus or B. bufo gargarizans). As a national second-class and self-developed TCM preparation, it is widely used to treat human colorectal cancer (CRC), yet its substantial toxic side effects, such as vascular irritation reaction, drug fever, and allergic reactions, pose a considerable challenge during clinical use. In-depth research on the material basis of HCS' traditional efficacy is essential for developing more effective and less toxic medicine. Identifying the main components responsible for the efficacy of HCS is crucial in determining its material basis and explaining any adverse reactions. PurposeThe purpose of this study is to clarify the main active components of HCS as a growth inhibitory agent against CRC HCT116 cells and to further understand the material basis of its anti-CRC properties. MethodsSulforhodamine B (SRB) assay was used to screen the compound with strong inhibitory activity against HCT116 cells from 44 compounds isolated from aqueous extracts of toad skin (B. melanosticus). The screened compounds as standard and MeOH extracts from HCS were determined by HPLC coupled with triple quadrupole mass spectrometry. The partial MeOH extract was subjected to HPLC system using solvents in a gradient of increasing polarity, yielding 12 fractions in total (HCS1 to HCS12). Besides, the screened active compounds were knocked out from HCS to obtain a fraction as a negative control, and the knocked-out components were collected as one fraction using semi-preparative HPLC. Those fractions were submitted to HPLC-MS/MS or HPLC-DAD system to determine the presence or absence of the screened compounds. In addition, cytotoxicity of those prepared fractions against CRC HCT116 cells was evaluated to determine the effect of the screened compounds in the anti-CRC of HCS. ResultsSix compounds, namely arenobufagin, telocinobufagin, gamabufalin, hellebrigenin, 19-hydroxylbufagin and argentinogenin, were screened for their inhibitory activity (with an IC50 value of less than 400 nM), and were present in HCS at a total-mass-percentage of 1.35×10−3 % in HCS MeOH extract. In addition, those 6 compounds were mainly distributed in HCS8, which also showed the strongest inhibitory activity against HCT116 cells. And the results of multicomponent knockout assay indicated the negative control lost its anti-CRC activity significantly, while the activity can be restored after those 6 compounds were added to the negative control. ConclusionsThose six bufadienolides derivatives are responsible for the anti-CRC HCT116 cells activity of HCS, and are the main components of HCS on anti-CRC. Through this research, we can more clearly understand the main anti-CRC active components in HCS, and the work also lays the foundation for the development of more effective and less toxic medicine from HCS.

CaERF1- mediated ABA signal positively regulates camptothecin biosynthesis by activating the iridoid pathway in Camptotheca acuminata

Camptotheca acuminata is one of the primary sources of camptothecin (CPT), which is widely used in the treatment of human malignancies because of its inhibitory activity against DNA topoisomerase I. Although several transcription factors have been identified for regulating CPT biosynthesis in other species, such as Ophiorrhiza pumila, the specific regulatory components controlling CPT biosynthesis in C. acuminata have yet to be definitively determined. In this study, CaERF1, an DREB subfamily of the APETALA2/ethylene response factors (AP2ERFs), was identified in C. acuminata. The transient overexpression and silencing of CaERF1 in C. acuminata leaves confirmed that it positively regulates the accumulation of CPT by inducing the expression of CaCYC1 and CaG8O in the iridoid pathway. Results of transient transcriptional activity assay and yeast one-hybrid assays have showed that CaERF1 transcriptionally activates the expression of CaCYC1 and CaG8O by binding to RAA and CEI elements in the promoter regions of these two genes. Furthermore, the expression of CaCYC1 and CaG8O in CaERF1-silenced leaves was less sensitive to ABA treatment, indicating that CaERF1 is a crucial component involved in ABA-regulated CPT biosynthesis in C. acuminata.

Thymol-loaded liposomes effectively induced apoptosis and decreased EGFR expression in colorectal cancer cells.

Colorectal cancer (CRC) is one of the most common and deadly cancers worldwide. Different factors, such as environmental and genetic factors and lifestyle, affect it. Owing to the presence of phenolic, alkaloid, antioxidant, and terpenoid compounds, herbal compounds can be effective in the treatment of various cancers. Thymol is a natural monoterpene phenol that is abundant in some plants and exerts several biological effects. The aim of this study was to investigate the apoptotic, anti-proliferative effect and EGFR gene expression under the influence of thymol-loaded nanoliposome in SW84 and SW111 cell lines derived from colorectal cancer. The lipid thin-film hydration method was used to synthesize thymol-loaded liposomes, and their characterization was performed using TEM, DLS, and HPLC analyses. SW84 and SW1111 cells were treated with thymol- and thymol-loaded liposomes at different doses, the inhibition of cell proliferation was evaluated using an MTT assay, the rate of apoptosis induction was assessed using flow cytometry, and EGFR gene expression was measured using real-time PCR. The nanoparticles produced were spherical, uniform, and 200 ± 10nm in size. HPLC analysis showed that approximately 98% thymol was loaded into the nanoliposome. The results of the MTT assay showed that thymol and thymol-nanoliposomes decreased the proliferation of SW84 and SW1111 cells in a concentration-dependent manner. The IC50 of thymol and thymol-nanoliposomes were 18 and 14.2µg/ml for the SW48 cell line (P = 0.04) and 10.5 and 6.4µg/ml for the SW1116 cell line (P = 0.001). Thymol-nanoliposomes significantly inhibited the proliferation of cancer cells compared to free thymol. Flow cytometry showed an increase in the percentage of apoptotic cells, especially in the thymol-nanoliposome group in the treated cells. Real-time PCR results also showed that thymol and thymol-nanoliposome both caused a decrease in the expression of EGFR genes in both cell lines, but this effect of decreasing gene expression was significantly higher in the thymol-nanoliposome group. Our results showed that thymol-nanoliposomes reduced proliferation, increased apoptosis, and decreased EGFR expression in colorectal cancer-derived cell lines.

Multi-Step Maceration Extraction and Pharmacological Evaluation of Abelmoschus manihot (L.) Medik

A study has been conducted on the phytochemical profile and pharmacological activity of Gedi (Abelmoschus manihot (L.) Medik) using a multistage maceration method. The results of the phytochemical screening test showed that the crude extract of Gedi leaves gave a positive reaction to the presence of alkaloids, tannins, phenols, flavonoids, unsaturated steroids and saponins. The total phenolic analysis showed that the total phenolic concentrations of Gedi leaf extracts in methanol, ethyl acetate, and hexane extracts were 1.70, 1.44, and 0.85mg GAE/g extract, respectively. The results of the antioxidant activity assays obtained an IC50 value for methanol, ethyl acetate, and hexane extracts of Gedi leaves, which were 40.77, 155.50, and 25.84mg/L, respectively. The antimicrobial activity results showed that all three Gedi leaf extracts had activity in inhibiting microbial growth. The inhibitory values for methanol, ethyl acetate, and hexane extracts of Gedi leaves were 7.0, 13.5, and 9.5mm, respectively, for Escherichia coli, and 21.5, 15.0, and 13.0mm, respectively, for Bacillus. The toxicity test results showed that the LC50 values of Gedi leaf extracts for methanol, ethyl acetate, and hexane extracts were 500 ppm, 736ppm, and 2249ppm, respectively.

Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

Integration of metabolomics and network pharmacology technology to explain the effect mechanisms of Danggui Buxue decoction in vascular dementia.

Danggui Buxue decoction (DBD) is a traditional Chinese medicine herbal decoction that has a good therapeutic effect on vascular dementia (VaD). However, its pharmacodynamic substances and underlying mechanisms are ambiguous. The work aimed to decipher the pharmacodynamic substances and molecular mechanisms of DBD against VaD rats based on gas chromatography-mass spectrometry metabonomics, network pharmacology, molecular docking, and experimental verification. The results indicated that DBD significantly improved the learning abilities and cognitive impairment in the VaD rat model. Integration analysis of the metabolomics and network pharmacology approach revealed that DBD might primarily affect arachidonic acid (AA) and inositol phosphate metabolic pathways by regulating the platelet activation signaling pathways. Six core targets (TNF [tumor necrosis factor], IL-6 [interleukin 6], PTGS2 [prostaglandin-endoperoxide synthase 2], MAPK1, MAPK3, and TP53) in the platelet activation signaling pathways also had a good affinity to seven main active components (saponins, organic acids, flavonoids, and phthalides) of DBD through the verification of molecular docking. Enzyme-linked immunosorbent assay results (ELISA) showed that the levels of TNF, IL-6, PTGS2, thromboxane B2, and caspase-3 in the platelet activation signaling pathway can be regulated by DBD. Our results indicated that DBD treated VaD mainly by modulating the platelet activation signaling pathway, and AA and inositol phosphate metabolism.

Investigation of DNA damage and inflammatory marker profile in patients after bariatric surgery

Abstract
 Purpose: Obesity is a significant risk factor in the development of many serious diseases. The most prominent ones among them are diabetes and coronary heart disease. Elevated blood sugar levels in obese individuals lead to increased susceptibility to infections due to the suppression of the immune response. Moreover, fatty skin folds can cause topical infections, ulcers, and delayed/impaired wound healing. Therefore, inflammatory and pro-inflammatory markers in the serum have gained importance in diseases such as diabetes and obesity. Furthermore, in obesity, reactive oxygen species (ROS) lead to DNA damage. 8-oxo-dG, which is the primary product of DNA oxidation, can be detected in the serum, saliva, and urine, making it an ideal biological marker for DNA damage in large population-based studies. The Comet assay analysis is a method used to demonstrate the double-strand breaks in DNA. Induction of γ-H2AX in tissue indicates the initiation of a well-regulated mechanism to reverse double-strand breaks in DNA. Potential benefits of monitoring the genomic health in obesity include creating a sense of urgency for personalized intervention measures and evaluating their progress. DNA damage in obesity is believed to be a reversible condition. Chronic inflammation is an etiological factor known to support DNA damage and neoplastic transformations in cells. Cytokines secreted from adipose tissue, especially TNF-alpha, IL-6, and IL-1β, promote the accumulation of various cells, including neutrophils, macrophages, and dendritic cells, and it indicates the initiation of an inflammatory process. In this project, it was aimed to investigate possible changes in inflammation markers and DNA damage in individuals undergoing bariatric surgery, who were beginning to improve endocrine and metabolic syndrome markers. 
 Materials and methods: For this purpose, blood and urine samples were collected from 45 obese patients, who had undergone bariatric surgery and 45 healthy volunteers matched for age and gender. The levels of inflammatory markers (IL-1β, IL-6, IL-8, and TNF-α) and the DNA damage marker γ-H2AX in serum, as well as the amount of 8-oxo-dG in urine, were determined using ELISA. Additionally, the percentage of DNA damage was determined using the Comet assay analysis. 
 Results: Weight control achieved through bariatric surgery and the subsequent reduction in fat tissue resulted in a significant decrease in the levels of γ-H2AX and 8-oxo-dG, as well as a parallel significant reduction in the percentage of DNA damage in the Comet assay results. The significant decrease in inflammatory markers IL-1β, IL-6, IL-8, and TNF-α levels indicated that bariatric surgery also affects inflammation indirectly. 
 Conclusion: Although there are numerous studies in the literature on individual parameters related to DNA damage in various diseases and obesity, it is believed that the present study determining DNA damage, oxidation, and repair mechanisms simultaneously with inflammatory marker levels serves as a guiding example for comparing genomic health and stability in pre-obese, post-obese, and non-obese individuals.

The antimicrobial and antibiofilm potential of the Piper nigrum L. essential oil: in vitro, in situ, and in silico study

The modern era has raised awareness of healthier lifestyles and food safety, creating new goals for scientists and industry. Here, pathogen microorganisms represent one of the difficulties in ensuring food quality. Microorganism’s resistance to broad-spectrum antibiotics has increased, especially with the planktonic cells' ability to switch into sessile mode and form biofilms. In the endeavor for efficient natural food preservatives, essential oils represent one of the possible solutions for controlling food spoilage, due to their recognized antimicrobial properties. In this study, the assessment of the chemical profile of Piper nigrum L. essential oil (PNEO) was performed. The GC-MS analysis identified (E)-caryophyllene, limonene, and sabinene as prevalent constituents of PNEO, which prompted further antimicrobial and antibiofilm investigations. The minimal inhibitory concentration (MIC) assays revealed that PNEO generally expressed better antimicrobial properties in comparison to the common antibiotics. Moreover, the in situ antimicrobial analysis uncovered the ability of the PNEO’s vapor phase to inhibit the growth of L. monocytogenes, S. aureus, and H. influenzae on selected food models, exposing PNEO as a promising preservation agent. Antibiofilm effects of PNEO were evaluated against biofilm-forming P. fluorescens and S. maltophilia employing MIC assays, crystal violet assays, MALDI-TOF MS analysis, and molecular docking. The obtained results in MIC and crystal violet assays indicated significant antibiofilm activity of PNEO. The MALDI-TOF MS analysis revealed that PNEO altered the protein profiles of the investigated bacteria on stainless steel and glass surfaces, especially in the case of S. maltophilia. In silico antibiofilm investigations indicated a potential involvement of the PNEO components in the interference of quorum-sensing and drug-resistance mechanisms. These findings underline the antimicrobial and antibiofilm virtues of PNEO, indicating its promising usage in food preservation.

A response surface model to examine the reactive red 239 sorption behaviors on Rhizoclonium hieroglyphicum: isotherms, kinetics, thermodynamics and toxicity analyses.

The present study is an attempt to investigate the potentiality of Rhizoclonium hieroglyphicum in the removal of reactive red 239 (RR239) from aqueous solution and to assess the toxicity of the treated dye solution. Optimisation of the process variables namely dye and biosorbent concentrations, pH, temperature and incubation time for RR239 removal was performed using Response Surface Methodology (RSM) assisted Box Behnken Design (BBD) model. The recycling and regeneration efficiency of the dye adsorbed alga was evaluated using different eluents under optimized conditions. Further to understand the adsorption mechanism, isotherms, kinetics and thermodynamic studies were performed. UV-vis and FT-IR spectroscopy was employed to confirm the interaction between the adsorbate and biosorbent. The nature of the treated dye solution was assessed using phyto, microbial and brine shrimp toxicity studies. On the basis of quadratic polynomial equation and response surfaces given by RSM, 90% decolorization of RR239 was recorded at room temperature under specified optimal conditions (300mg/L of dye, 500mg/L of biosorbent, pH 8 and 72h of contact time). Desorption experiments demonstrated 88% of RR239 recovery using 0.1N acetic acid as an eluent and 81% of dye removal in regeneration studies. The data closely aligned with Freundlich isotherm (R2 - 0.98) and pseudo-second-order kinetic model (R2 - 0.9671). Thermodynamic analysis revealed that the process of adsorption was endothermic, spontaneous, and favorable. UV-Vis and FT-IR analyses provided evidence for adsorbate-biosorbent interaction, substantiating the process of decolorization. In addition, the results of phyto, microbial and brine shrimp toxicity assays consistently confirmed the non-toxic nature of the treated dye. Thus, the study demonstrated that R. hieroglyphicum can act as a potent bioremediation agent in alleviating the environmental repercussions of textile dyeing processes.

An Anti-CD7 Antibody-Drug Conjugate Target Showing Potent Antitumor Activity for T-Lymphoblastic Leukemia (T-ALL).

Acute T-lymphoblastic leukemia (T-ALL) is a type of leukemia that can occur in both pediatric and adult populations. Compared to acute B-cell lymphoblastic leukemia (B-ALL), patients with T-cell T-ALL have a poorer therapeutic efficacy. In this study, a novel anti-CD7 antibody-drug conjugate (ADC, J87-Dxd) was successfully generated and used for T-ALL treatment. Firstly, to obtain anti-CD7 mAbs, we expressed and purified the CD7 protein extracellular domain. Utilizing hybridoma technology, we obtained three anti-CD7 mAbs (J87, G73 and A15) with a high affinity for CD7. Both the results of immunofluorescence and Biacore assay indicated that J87 (KD = 1.54 × 10-10 M) had the highest affinity among the three anti-CD7 mAbs. In addition, an internalization assay showed the internalization level of J87 to be higher than that of the other two mAbs. Next, we successfully generated the anti-CD7 ADC (J87-Dxd) by conjugating DXd to J87 via a cleavable maleimide-GGFG peptide linker. J87-Dxd also possessed the ability to recognize and bind CD7. Using J87-Dxd to treat T-ALL cells (Jurkat and CCRF-CEM), we observed that J87-Dxd bound to CD7 was internalized into T-ALL cells. Moreover, J87-Dxd treatment significantly induced the apoptosis of Jurkat and CCRF-CEM cells. The IC50 (half-maximal inhibitory concentration) value of J87-Dxd against CCRF-CEM obtained by CCK-8 assay was 6.3 nM. Finally, to assess the antitumor efficacy of a J87-Dxd in vivo, we established T-ALL mouse models and treated mice with J87-Dxd or J87. The results showed that on day 24 after tumor inoculation, all mice treated with J87 or PBS died, whereas the survival rate of mice treated with J87-Dxd was 80%. H&E staining showed no significant organic changes in the heart, liver, spleen, lungs and kidneys of all mice. In summary, we demonstrated that the novel anti-CD7 ADC (J87-Dxd) had a potent and selective effect against CD7-expressing T-All cells both in vitro and in vivo, and could thus be expected to be further developed as a new drug for the treatment of T-ALL or other CD7-expression tumors.

AcMYB10 Involved in Anthocyanin Regulation of 'Hongyang' Kiwifruit Induced via Fruit Bagging and High-Postharvest-Temperature Treatments.

Light and temperature are key factors influencing the accumulation of anthocyanin in fruit crops. To assess the effects of fruit bagging during development and high post-ripening temperature on 'Hongyang' kiwifruit, we compared the pigmentation phenotypes and expression levels of anthocyanin-related genes between bagged and unbagged treatments, and between 25 °C and 37 °C postharvest storage temperatures. Both the bagging and 25 °C treatments showed better pigmentation phenotypes with higher anthocyanin concentrations. The results of the qRT-PCR analysis revealed that the gene expression levels of LDOX (leucoanthocyanidin dioxygenase), F3GT (UDP-flavonoid 3-O-glycosyltransferase ), AcMYB10, and AcbHLH42 were strongly correlated and upregulated by both the bagging treatment and 25 °C storage. The results of bimolecular fluorescence complementation and luciferase complementation imaging assays indicated an interaction between AcMYB10 and AcbHLH42 in plant cells, whereas the results of a yeast one-hybrid assay further demonstrated that AcMYB10 activated the promoters of AcLODX and AcF3GT. These results strongly suggest that enhanced anthocyanin synthesis is caused by the promoted expression of AcLODX and AcF3GT, regulated by the complex formed by AcMYB10-AcbHLH42.

Leech extract alleviates idiopathic pulmonary fibrosis by TGF-β1/Smad3 signaling pathway

Ethnopharmacological relevanceLeech, as a traditional Chinese medicine for the treatment of blood circulation and blood stasis, was also widely used to cure pulmonary fibrosis in China. In clinical practice, some traditional Chinese medicine preparation such as Shui Zhi Xuan Bi Hua Xian Tang and Shui Zhi Tong Luo Capsule composed of leech, could improve the clinical symptoms and pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the material basis of the leech in the treatment of IPF were not yet clear. Aim of the studyScreen out the components of leech that have the anti-pulmonary fibrosis effects, and further explore the therapeutic mechanism of the active components. Materials and methodsIn this study, the different molecular weight components of leech extract samples were prepared using the semi-permeable membranes with different pore sizes. The therapeutic effects of the leech extract groups with molecular weight greater than 10 KDa (>10 KDa group), between 3 KDa and 10 KDa (3–10 KDa group), and less than 3 KDa (<3 KDa group) on pulmonary fibrosis were firstly investigated by cell proliferation and cytotoxicity assay (MTT), cell wound healing assay, immunofluorescence staining (IF) and Western blot (WB) assay through the TGF-β1-induced fibroblast cell model. Then bleomycin-induced pulmonary fibrosis (BML-induced PF) mouse model was constructed to investigate the pharmacological activities of the active component group of leech extract in vivo. Pathological changes of the mouse lung were observed by hematoxylin-eosin staining (H&E) and Masson's trichrome staining (Masson). The hydroxyproline (HYP) content of lung tissues was quantified by HYP detection kit. The levels of extracellular matrix-related fibronectin (FN) and collagen type Ⅰ (Collagen Ⅰ), pyruvate kinase M2 (PKM2) monomer and Smad7 protein were determined via WB method. PKM2 and Smad7 protein were further characterized by IF assays. ResultsUsing TGF-β1-induced HFL1 cell line as a PF cell model, the in vitro results demonstrated that the >10 KDa group could significantly inhibited the cell proliferation and migration, downregulated the expression level of cytoskeletal protein vimentin and α-smooth muscle actin (α-SMA), and reduced the deposition of FN and Collagen Ⅰ. In the BML-induced PF mouse model, the >10 KDa group significantly reduced the content of HYP, downregulated the expression levels of FN and Collagen Ⅰ in lung tissues, and delayed the pathological changes of lung tissue structure. The results of WB and IF assays further indicated that the >10 KDa group could up-regulate the expression level of PKM2 monomer and Smad7 protein in the cellular level, thereby delaying the progression of pulmonary fibrosis. ConclusionsOur study revealed that the >10 KDa group was the main material basis of the leech extract that inhibited pulmonary fibrosis through TGF-β1/Smad3 signaling pathway.

Syncephalastrum massiliense sp. nov. and Syncephalastrum timoneanum sp. nov. Isolated from Clinical Samples.

Mucormycosis is known to be a rare opportunistic infection caused by fungal organisms belonging to the Mucorales order, which includes the Syncephalastrum species. These moulds are rarely involved in clinical diseases and are generally seen as contaminants in clinical laboratories. However, in recent years, case reports of human infections due to Syncephalastrum have increased, especially in immunocompromised hosts. In this study, we described two new Syncephalastrum species, which were isolated from human nails and sputum samples from two different patients. We used several methods for genomic and phenotypic characterisation. The phenotypic analysis relied on the morphological features, analysed both by optical and scanning electron microscopy. We used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, energy-dispersive X-ray spectroscopy, and BiologTM technology to characterise the proteomic, chemical mapping, and carbon source assimilation profiles, respectively. The genomic analysis relied on a multilocus DNA sequence analysis of the rRNA internal transcribed spacers and D1/D2 large subunit domains, fragments of the translation elongation factor-1 alpha, and the β-tubulin genes. The two novel species in the genus Syncephalastrum, namely S. massiliense PMMF0073 and S. timoneanum PMMF0107, presented a similar morphology: irregular branched and aseptate hyphae with ribbon-like aspects and terminal vesicles at the apices all surrounded by cylindrical merosporangia. However, each species displayed distinct phenotypic and genotypic features. For example, S. timoneanum PMMF0107 was able to assimilate more carbon sources than S. massiliense PMMF0073, such as adonitol, α-methyl-D-glucoside, trehalose, turanose, succinic acid mono-methyl ester, and alaninamide. The polyphasic approach, combining the results of complementary phenotypic and genomic assays, was instrumental for describing and characterising these two new Syncephalastrum species.

HvGSK1.1 Controls Salt Tolerance and Yield through the Brassinosteroid Signaling Pathway in Barley.

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits.

Assessment of the regional composition of juvenile chum salmon mixed aggregations in the Sea of Okhotsk based on results of genetic assay

Genetic differentiation of chum salmon in the basin of the Sea of Okhotsk is demonstrated. Eight microsatellite loci (Ssa20.19, One101, Oke3, Oki1b, Oki23, Ogo2G, Oke11, Ots102) were used as genetic markers. Four original genetic regions of chum salmon reproduction have been figured out: West Kamchatka and northern part of the continental coast of the Sea of Okhotsk, Sakhalin, Ithurup (the Kuril Islands), the Amur basin, where the part of juvenile fish was found as a result of genetic identification in the mixed aggregations in the early period of marine feeding. The resolution ability of the reference database was within the limits 74.9–94.6 %. It was demonstrated based on results of the genetic identification, that juveniles from West Kamchatka and northern part of the continental coast of the Sea of Okhotsk dominate in the northern part of the Sea of Okhotsk. Majority of individuals examined in the central zone of the survey area (54 %) was from the Kuril Islands. A number of juvenile chum salmon from the Amur River basin and Sakhalin were recorded at the stations in vicinity of Sakhalin coast.

An international inter-laboratory study to compare digital PCR with ISO standardized qPCR assays for the detection of norovirus GI and GII in oyster tissue

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Development of a tag-free plant-made interferon gamma production system with improved therapeutic efficacy against viruses.

Plants offer a promising platform for cost-effective production of biologically active therapeutic glycoproteins. In previous studies, we have developed a plant expression system based on Bamboo mosaic virus (BaMV) by incorporating secretory signals and an affinity tag, which resulted in notably enhanced yields of soluble and secreted fusion glycoproteins (FGs) in Nicotiana benthamiana. However, the presence of fusion tags on recombinant glycoproteins is undesirable for biomedical applications. This study aimed to develop a refined expression system that can efficiently produce tag-free glycoproteins in plants, with enhanced efficacy of mature interferon gamma (mIFNγ) against viruses. To accommodate the specific requirement of different target proteins, three enzymatically or chemically cleavable linkers were provided in this renovated BaMV-based expression system. We demonstrated that Tobacco etch virus (TEV) protease could process the specific cleavage site (LTEV) of the fusion protein, designated as SSExtHis(SP)10LTEV-mIFNγ, with optimal efficiency under biocompatible conditions to generate tag-free mIFNγ glycoproteins. The TEV protease and secretory-affinity tag could be effectively removed from the target mIFNγ glycoproteins through Ni2+-NTA chromatography. In addition, the result of an antiviral assay showed that the tag-free mIFNγ glycoproteins exhibited enhanced biological properties against Sindbis virus, with comparable antiviral activity of the commercialized HEK293-expressed hIFNγ. Thus, the improved BaMV-based expression system developed in this study may provide an alternative strategy for producing tag-free therapeutic glycoproteins intended for biomedical applications.