Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer.

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

Biocompatible Ferrofluid-Based Millirobot for Tumor Photothermal Therapy in Near-Infrared-II Window.

Ferrofluidic robots with excellent deformability and controllability have been intensively studied recently. However, most of these studies are in vitro and the use of ferrofluids for in vivo medicinal applications remains a big challenge. The application of ferrofluidic robots to the body requires the solution of many key problems. In this study, biocompatibility, controllability, and tumor-killing efficacy are considered when creating a ferrofluid-based millirobot for in vivo tumor-targeted therapy. For biocompatibility problems, corn oil is used specifically for the ferrofluid robot. In addition, a control system is built that enables a 3D magnetic drive to be implemented in complex biological media. Using the photothermal conversion property of 1064 nm, the ferrofluid robot can kill tumor cells in vitro; inhibit tumor volume, destroy the tumor interstitium, increase tumor cell apoptosis, and inhibit tumor cell proliferation in vivo. This study provides a reference for ferrofluid-based millirobots to achieve targeted therapies in vivo.

Goat milk powder supplemented with branched-chain fatty acid: influence on quality and microstructure.

BACKGROUND: Branched-chain fatty acid (BCFA) is effective in preventing and helping to treat neonatal necrotizing enterocolitis. It is essential to supplement goat-milk powder for formula-fed preterm infants with BCFA. In this study, the quality and microstructures of milk powders supplemented with different concentrations of BCFA were evaluated, using goat milk powder without BCFA as the control group (CG). RESULTS: In comparison with the CG, goat milk powder supplemented with BCFA exhibited smaller fat globules and a significant drop in overall particle size. During 16 weeks of storage, BCFA-supplemented groups showed suitable moisture content and viscosity and good solubility. The BCFA also helped reduce the number of folds on the surface of the milk powder particles. CONCLUSION: The findings of this study indicate that goat milk powders with BCFA exhibit differences in quality and microstructure in comparison with ordinary goat milk powder, which is relevant for the future development and application of BCFA in foods. © 2022 Society of Chemical Industry.

Self-assembled nano-photosensitizer for targeted, activatable, and biosafe cancer phototheranostics.

Cancer treatment currently still faces crucial challenges in therapeutic effectiveness, precision, and complexity. Photodynamic therapy (PDT) as a non-invasive tactic has earned widespread popularity for its excellent therapeutic output, flexibility, and restrained toxicity. Nonetheless, drawbacks, including low efficiency, poor cancer specificity, and limited therapeutic depth, remain considerable during the cancer treatment. Although great effort has been made to improve the performance, the overall efficiency and biosafety are still ambiguous and unable to meet urgent clinical needs. Herein, this study integrates merits from previous PDT strategies and develops a cancer-targeting, activatable, biosafe photosensitizer. Owing to excellent self-assembly ability, this photosensitizer can be conveniently prepared as multifunctional nano-photosensitizers, namely MBNPs, and applied to in vivo cancer phototheranostics in "all-in-one" mode. This study successfully verifies the mechanism of MBNPs, then deploys them to cell-based and in vivo cancer PDT. Based on the unique cancer microenvironment, MBNPs achieve precise distribution, accumulation, and activation towards the tumor, releasing methylene blue as a potent photosensitizer for phototherapy. The PDT outcome demonstrates MBNPs' superior cancer specificity, remarkable PDT efficacy, and negligible toxicity. Meanwhile, in vivo NIR fluorescence and photoacoustic imaging have been utilized to guide the PDT treatment synergistically. Additionally, the biosafety of the MBNPs-based PDT treatment is ensured, thus providing potential for future clinical studies.

Extraction optimization of polyphenols from fruits of Pyracantha fortuneana (Maxim.) Li by ultrasonic assistant method and their antibacterial activity.

The present study was designed to utilise ultrasound assistance technology to optimize the extraction conditions of polyphenols, and identify their antibacterial activity from fruits of Pyracantha fortuneana (Maxim.) Li, facilitated by the use of orthogonal experiment methodology. A four factors and three levels of orthogonal design was carried out to elucidate the effect of ethanol concentration, solvent-to-solid ratio, ultrasonic temperature and time on the yields of the polyphenols. The results showed that the optimal conditions were as follows: ethanol concentration 70%, solvent-to-solid ratio 70:1, ultrasonic temperature 30°C, and ultrasonic time 40 min, the maximum polyphenol yield was 5.58mg/g under the optimum extraction condition. The extracted hydro-alcohol polyphenols showed the excellent antibacterial potential to both Staphylococcus aureus and Escherichia coli with the minimum inhibitory concentration (MIC) 10 mg/ml and 20 mg/ml, and the corresponding diameter of inhibition zone (DIZ) was 7.4 and 6.8 mm, respectively. The results indicated the ability of ultrasound assistance technology to obtain polyphenols from fruits. Furthermore, present results highlighted that fruits of Pyracantha fortuneana (Maxim.) Li are a potential natural source of bioactive compounds with strong antibacterial activity. These compounds could be considered for potential application in nutraceutical and functional foods ingredient or pharmaceutical applications.

Scolopendra subspinipes mutilans L. Koch Ameliorates Rheumatic Heart Disease by Affecting Relative Percentages of CD4+CD25+FoxP3 Treg and CD4+IL17 T Cells.

(Scolopendra subspinipes mutilans L. Koch. (SSLK) helps reduce the risk of coronary heart disease (CHD) but its effects on rheumatic heart disease (RHD) patients remain unclear. 80 RHD patients were recruited and randomly assigned into SG (to receive SSLK treatment) and CG (to receive placebo) groups, and the intervention lasted for 3 months. The following cardiac indexes were measured, including mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), blood lactate, fatigue, shortness of breath, palpitation, and chest pain. ELISA kits were used to analyze creatine kinase isoenzyme (CK-MB), serum troponin T (cTnT), CRP, IL-1ß, IL-6, and TNF-α, malondialdehyde (MDA), and superoxide dismutase (SOD). Relative percentages of CD4+CD25+FoxP3 regulatory (Treg) and CD4+IL-17 T cells were measured using flow cytometry. After 3-month therapy, SSLK intervention improved MAP, HR, CVP, fatigue, palpitation, and shortness breath of CHD patients, reduced the levels of blood lactate, CK-MB, cTnT, CRP, IL-1ß, IL-6, TNF-α, MDA, and increased SOD level (p < 0.05). Meanwhile, SSLK treatment increased the percentages of CD4+CD25+FoxP3 Treg cells and reduced relative percentages of CD4+IL-17 T cells in a dose-dependent way (p < 0.05). Relative percentage of CD4+CD25+FoxP3 Treg cells had negative relationship while CD4+IL17 T cells had positive relationship with CK-MB, cTnT, CRP, and TNF-a (p < 0.01). SSLK ameliorated RHD by affecting the balance of CD4+CD25+FoxP3 Treg and CD4+IL17 T cells.

Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells.

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Two proprotein convertase subtilisin/kexin type 9 (PCSK9) paralogs from the tropical sea cucumber (Stichopus monotuberculatus): Molecular characterization and inducible expression with immune challenge.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a multifunctional protein that widely exists in eukaryotic species. In this study, two PCSK9 paralogs, named StmPCSK9-1 and StmPCSK9-2, were identified from the tropical sea cucumber (Stichopus monotuberculatus). The cDNAs of StmPCSK9-1 and StmPCSK9-2 are 1330 kb and 1508 kb in size, respectively. The open reading frames (ORF) for StmPCSK9-1 and StmPCSK9-2 cDNAs are 1128 and 1167 bp in length, encoding the proteins of 375 and 388 amino acids with the deduced molecular weights of 38.76 and 41.07 kDa, respectively. In accord with other members in PCSK9 family, the two StmPCSK9 paralogs possessed the inhibitor_I9 and peptidase_S8 functional domains, seven active sites, a catalytic triad and two calcium binding sites. For the gene structure, the splicing of the two StmPCSK9 paralogs was relatively conserved. In addition, the mRNA expression of StmPCSK9-1 and StmPCSK9-2 was only detected in the sea cucumber intestine and coelomocytes, and the expression levels of both the two StmPCSK9 paralogs were higher in intestine. Moreover, StmPCSK9-2 was found to be a cytoplasm protein without signal peptide, and show no response to the immune challenge. On the contrary, StmPCSK9-1 was a secreted protein and the transcriptional expression of StmPCSK9-1 was significantly up-regulated by lipopolysaccharides (LPS) treatment and slightly down-regulated by polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge in in vitro experiments performed in the cultural primary coelomocytes, suggesting that the StmPCSK9-1 may play critical roles in the innate immune defense of sea cucumber, S. monotuberculatus, against bacterial and/or viral infections.

Physiological and proteomic analysis of selenium-mediated tolerance to Cd stress in cucumber (Cucumis sativus L.).

Selenium can mitigate cadmium toxicity in plants. However, the mechanism of this alleviation has not been fully understood. In the present study, the role of Se in inducing tolerance to Cd stress in cucumber was elucidated. Results showed that Se significantly alleviated Cd-induced growth inhibition, reduced Cd concentration, increased SPAD value and improved photosynthetic performance. Through proteomic analysis by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry, 26 protein spots were identified, which were significantly influenced by Cd stress and/or Se application. Among these proteins, the abundance of 21 spots (10 in leaves and 11 in roots) were repressed in Cd-treated and up-accumulated or no-changed in Cd+Se-treated cucumber. These altered proteins were involved in the response to stress, metabolism, photosynthesis and storage, they were including glutathione S-transferase F8, heat shock protein STI-like, peroxidase, ascorbate oxidase, fructose-bisphosphate aldolase 2, NiR, Rieske type ion sulfur subunit and PsbP domain-containing protein 6. Furthermore, we identified five proteins with an increase in relative abundance after Cd treatment, they were involved in the functional groups active in response to stress and transport. The present study provided novel insights into Se-mediated tolerance of cucumber seedlings against Cd toxicity at the proteome level.

Target identification of natural and traditional medicines with quantitative chemical proteomics approaches.

Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines.

The functional gene composition and metabolic potential of coral-associated microbial communities.

The phylogenetic diversity of coral-associated microbes has been extensively examined, but some contention remains regarding whether coral-associated microbial communities are species-specific or site-specific. It is suggested that corals may associate with microbes in terms of function, although little is known about the differences in coral-associated microbial functional gene composition and metabolic potential among coral species. Here, 16S rRNA Illumina sequencing and functional gene array (GeoChip 5.0) were used to assess coral-associated microbial communities. Our results indicate that both host species and environmental variables significantly correlate with shifts in the microbial community structure and functional potential. Functional genes related to key biogeochemical cycles including carbon, nitrogen, sulfur and phosphorus cycling, metal homeostasis, organic remediation, antibiotic resistance and secondary metabolism were shown to significantly vary between and among the four study corals (Galaxea astreata, Porites lutea, Porites andrewsi and Pavona decussata). Genes specific for anammox were also detected for the first time in the coral holobiont and positively correlated with ammonium. This study reveals that variability in the functional potential of coral-associated microbial communities is largely driven by changes in environmental factors and further demonstrates the importance of linking environmental parameters with genomic data in complex environmental systems.

Study on the changes of thrombosis-associated factors in patients with coronary heart disease of turbidity-phlegm blocking syndrome.

OBJECTIVE: To preliminarily explore the relationship between thrombosis and its associated factors in patients with coronary heart disease (CHD) of turbidity-phlegm blocking syndrome (TPB), and to study its acting mechanism. METHODS: Plasma levels of thrombosis-associated factors, including Von Willebrand factor: (vWF), D-dimer, and fibrinogen (Fg), in 85 patients of CHD with TPB, 93 with CHD of non-TPD, and 89 healthy persons were detected and compared. RESULTS: Levels of the three factors were increased in all the CHD: patients, and were higher in TPB patients than in non-TPB patients (P<0.01 or P<0.05). CONCLUSION: The TPB: syndrome in CHD patients was closely related to the blood coagulation-fibrinolytic system; they might be in pre-thrombosis state, and the plasma levels of vWF, D-dimer, and Fg could be taken as the objective indices for thrombosis differentiation of TPB syndrome in CHD patients.

[Clinical study of Wenxikeli treatment of viral myocarditis].

OBJECTIVE: To observe Wenxinkeli viral myocarditis clinical efficacy and safety. METHODS: 45 patients were randomly divided into two groups. Treatment group given Wenxinkeli (5 g, 3 times a day with warm water) treatment, the control group given adenosine triphosphate, coenzyme A, vitamin C, coenzyme Q10 and other nutrients cardiac drugs, treatment for 4 weeks. RESULTS: The treatment of clinical symptoms, signs and ECG improvement, restoration of enzyme than the control group, there was a significant difference (P < 0.05). CONCLUSIONS: Wenxinkeli ventricular premature treatment of viral myocarditis is effective, and no significant adverse reactions, safety, good.