Seamless phase 2/3 design for trials with multiple co-primary endpoints using Bayesian predictive power.

Seamless phase 2/3 design has become increasingly popular in clinical trials with a single endpoint. Trials that define success based on the achievement of all co-primary endpoints (CPEs) encounter the challenge of inflated type 2 error rates, often leading to an overly large sample size. To tackle this challenge, we introduced a seamless phase 2/3 design strategy that employs Bayesian predictive power (BPP) for futility monitoring and sample size re-estimation at interim analysis. The correlations among multiple CPEs are incorporated using a Dirichlet-multinomial distribution. An alternative approach based on conditional power (CP) was also discussed for comparison. A seamless phase 2/3 vaccine trial employing four binary endpoints under the non-inferior hypothesis serves as an example. Our results spotlight that, in scenarios with relatively small phase 2 sample sizes (e.g., 50 or 100 subjects), the BPP approach either outperforms or matches the CP approach in terms of overall power. Particularly, with n1 = 50 and ρ = 0, BPP showcases an overall power advantage over CP by as much as 8.54%. Furthermore, when the phase 2 stage enrolled more subjects (e.g., 150 or 200), especially with a phase 2 sample size of 200 and ρ = 0, the BPP approach evidences a peak difference of 5.76% in early stop probability over the CP approach, emphasizing its better efficiency in terminating futile trials. It's noteworthy that both BPP and CP methodologies maintained type 1 error rates under 2.5%. In conclusion, the integration of the Dirichlet-Multinominal model with the BPP approach offers improvement in certain scenarios over the CP approach for seamless phase 2/3 trials with multiple CPEs.

Microplastics induced apoptosis in macrophages by promoting ROS generation and altering metabolic profiles.

The ubiquitous presence of Microplastics (MPs) in various environments documented in recent years has recently raised significant concerns about their toxic effects. While macrophages serve as the first line of defense against toxic substances and pathogens, the impact and mechanisms of microplastics on these immune cells remain unclear. This study aims to explore whether MPs induce macrophage apoptosis through the promotion of reactive oxygen species (ROS) generation and alterations in metabolic profiles. The viability of RAW264.7 cells decreased as the concentration of 0.5 µm or 5 µm MPs ranged from 0.2 to 1.5 mg/mL, with a more pronounced effect observed in the 0.5 µm MPs group. Zebrafish exposed to 0.5 µm or 5 µm MPs at a concentration of 0.5 mg/mL exhibited decreased macrophage abundance and increased apoptosis, accompanied by alterations in the expression of inflammatory and apoptosis-related genes. While 0.5 µm MPs were observed to enter macrophages, 5 µm MPs only adhered to the cell membrane surface. Both particle sizes induced ROS generation and disrupted cellular metabolism in RAW264.7 cells. Notably, macrophages exhibited a more pronounced response to 0.5 µm MPs, characterized by heightened ROS generation, increased secretion of pro-inflammatory mediators, and a significant decrease in sphingolipid metabolism. These findings suggest that the adverse effects on macrophages are greater with 0.5 µm MPs compared to 5 µm MPs, possibly attributed to particle size effects. This study contributes additional evidence on the impact of MPs on human immune cells.

Phytochemicals in regulating PD-1/PD-L1 and immune checkpoint blockade therapy.

Clinical treatment and preclinical studies have highlighted the role of immune checkpoint blockade in cancer treatment. Research has been devoted to developing immune checkpoint inhibitors in combination with other drugs to achieve better efficacy or reduce adverse effects. Phytochemicals sourced from vegetables and fruits have demonstrated antiproliferative, proapoptotic, anti-migratory, and antiangiogenic effects against several cancers. Phytochemicals also modulate the tumor microenvironment such as T cells, regulatory T cells, and cytokines. Recently, several phytochemicals have been reported to modulate immune checkpoint proteins in in vivo or in vitro models. Phytochemicals decreased programmed cell death ligand-1 expression and synergized programmed cell death receptor 1 (PD-1) monoclonal antibody to suppress tumor growth. Combined administration of phytochemicals and PD-1 monoclonal antibody enhanced the tumor growth inhibition as well as CD4+ /CD8+ T-cell infiltration. In this review, we discuss immune checkpoint molecules as potential therapeutic targets of cancers. We further assess the impact of phytochemicals including carotenoids, polyphenols, saponins, and organosulfur compounds on cancer PD-1/programmed cell death ligand-1 immune checkpoint molecules and document their combination effects with immune checkpoint inhibitors on various malignancies.

ccTCM: A quantitative component and compound platform for promoting the research of traditional Chinese medicine.

Traditional Chinese medicine (TCM) databases play a vital role in bridging the gap between TCM and modern medicine, as well as in promoting the popularity of TCM. Elucidating the bioactive ingredients of Chinese medicinal materials is key to TCM modernization and new drug discovery. However, one drawback of current TCM databases is the lack of quantitative data on the constituents of Chinese medicinal materials. Herein, we present ccTCM, a web-based platform designed to provide a component and compound-content-based resource on TCM and analysis services for medical experts. In terms of design features, ccTCM combines resource distribution, similarity analysis, and molecular-mechanism analysis to accelerate the discovery of bioactive ingredients in TCM. ccTCM contains 273 Chinese medicinal materials commonly used in clinical settings, covering 29 functional classifications. By searching and comparing, we finally adopted 2043 studies, from which we collected the compounds contained in each TCM with content greater than 0.001 %, and a total of 1449 were extracted. Subsequently, we collected 40,767 compound-target pairs by integrating multiple databases. Taken together, ccTCM is a versatile platform that can be used by TCM scientists to perform scientific and clinical TCM studies based on quantified ingredients of Chinese medicinal materials. ccTCM is freely accessible at http://www.cctcm.org.cn.

Binding and neutralizing abilities of antibodies towards SARS-CoV-2 S2 domain.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants have been reported to be resistant to several neutralizing antibodies (NAbs) targeting Receptor Binding Domain (RBD) and N Terminal Domain (NTD) of spike (S) protein and thus inducing immune escape. However, fewer studies were carried out to investigate the neutralizing ability of S2-specific antibodies. In this research, 10 monoclonal antibodies (mAbs) targeting SARS-CoV-2 S2 subunit were generated from Coronavirus Disease 2019 (COVID-19) convalescent patients by phage display technology and molecular cloning technology. The binding activity of these S2-mAbs toward SARS-CoV-2 S, SARS-CoV-2 S2, SARS-CoV-2 RBD, SARS-CoV-2 NTD, severe acute respiratory syndrome coronavirus (SARS-CoV) S, SARS-CoV S2 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) S proteins were evaluated by enzyme-linked immunosorbent assay (ELISA). Their neutralizing potency toward SARS-CoV-2 wild-type (WT), B.1.1.7, B.1.351, P.1, B.1.617.2, B.1.1.1 and B.1.621 variants were determined by pseudo-virus-based neutralization assay. Results showed that S2E7-mAb had cross-activity to S or S2 proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, while with limited neutralizing activity to pseudo-viruses of SARS-CoV-2 WT and variants. It is undeniable that the binding and neutralizing activities of the S2-targeting mAbs are significantly weaker than the previously reported antibodies targeting RBD and NTD, but our study may provide some evidences for understanding immune protection and identifying targets for vaccine design based on the conserved S2 subunit.

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2.

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

Gut microbiota-mediated tributyltin-induced metabolic disorder in rats.

Tributyltin (TBT), an environmental pollutant widely used in antifouling coatings, can cause multiple-organ toxicity and gut microbiome dysbiosis in organisms, and can even cause changes in the host metabolomic profiles. However, little is known about the underlying effects and links of TBT-induced metabolic changes and gut microbiome dysbiosis. In this study, rats were exposed to TBT at a dose of 100 µg kg-1 body weight (BW) for 38 days, followed by multi-omics analysis, including microbiome, metabolomics, and metallomics. Results showed that TBT exposure reduced rat weight gain and decreased the serum triglyceride (TG) level. Metabolic analysis revealed that TBT fluctuated linoleic acid metabolism and glycerophospholipid metabolism in the liver; the tricarboxylic acid cycle (TCA cycle), nicotinate and nicotinamide metabolism, and arachidonic acid metabolism in serum; glycine, serine, and threonine metabolism, the one carbon pool by folate, nicotinate, and nicotinamide metabolism; and tryptophan metabolism in feces. Furthermore, TBT treatment dictated liver inflammation due to enhancing COX-2 expression by activating protein kinase R-like ER kinase (PERK) and C/EBP homologous protein (CHOP) to induce endoplasmic reticulum (ER) stress instead of stimulating arachidonic acid metabolism. Meanwhile, alteration of the intestinal flora [Acetivibrio]_ethanolgignens_group, Acetatifactor, Eisenbergiella, Lachnospiraceae_UCG-010, Enterococcus, Anaerovorax, and Bilophila under TBT exposure were found to be involved in further mediating liver inflammation, causing lipid metabolism abnormalities, such as TG, linoleic acid, and glycerophospholipids, and interfering with the energy supply process. Among these, [Acetivibrio]_ethanolgignens_group, Enterococcus, and Bilophila could be considered as potential biomarkers for TBT exposure based on receiver operator characteristic (ROC) curve analysis.

Development of Patient-Derived Human Monoclonal Antibodies Against Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus 2 for Coronavirus Disease 2019 Diagnosis.

The pandemic caused by emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a global public health threat. Illustrating human antibody responding to viral antigen could potentially provide valuable information for basic research and clinical diagnosis. The antibody can be used as a complement to the viral detection for the rapid diagnosis of infected patients. Compared with spike protein (SP), nucleocapsid protein (NP) is normally conserved and highly immunogenic in many coronavirus members. As a major antigen, NP is a potential target for the diagnosis of SARS-CoV-2 infection. Here, we constructed a combinatorial fragment of antigen-binding (Fab)antibody phage library based on peripheral blood-derived from five coronavirus disease 2019 (COVID-19) infected donors. From the library, 159 Fab antibodies were obtained and identified by panning with NP. Among them, 16 antibodies were evaluated for their binding properties and epitopes recognition. Among these 16 antibodies, two well-paired antibodies were finally screened out for SARS-CoV-2 diagnosis by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. Our works may provide a potential resource for the clinical diagnosis of SARS-CoV-2 infection.

Comparative effects of mercury chloride and methylmercury exposure on early neurodevelopment in zebrafish larvae.

Mercury (Hg) is a ubiquitous environmental toxicant with important public health implications. Hg causes neurotoxicity through astrocytes, Ca2+, neurotransmitters, mitochondrial damage, elevations of reactive oxygen species and post-translational modifications. However, the similarities and differences between the neurotoxic mechanisms caused by different chemical forms of Hg remain unclear. Zebrafish embryos were exposed to methylmercury (MeHgCl) or mercury chloride (HgCl2) (0, 4, 40, 400 nM) up for 96 h. HgCl2 exposure could significantly decrease survival rate, body length and eye size, delay the hatching period, induce tail bending and reduce the locomotor activity, and these effects were aggravated in the MeHgCl group. The compounds could increase the number of apoptotic cells in the brain and downregulate the expression of Shha, Ngn1 and Nrd, which contribute to early nervous development. The underlying mechanisms were investigated by metabolomics data. Galactose metabolism, tyrosine metabolism and starch and sucrose metabolism pathways were disturbed after HgCl2 or MeHgCl exposure. In addition, the levels of three neurotransmitters including tyrosine, dopamine and tryptophan were reduced after HgCl2 or MeHgCl exposure. Oxidative stress is related to metabolite changes, such as changes in the putrescine, niacinamide and uric acid contents in the HgCl2 group, and squalene in the MeHgCl group. These data indicated that downregulation of these genes and abnormal metabolic profile and pathways contribute to the neurotoxicity of HgCl2 and MeHgCl.

Synthesis of MoS2 nanosheets for mercury speciation analysis by HPLC-UV-HG-AFS.

Mercury species have aroused wide concern in the past several decades due to their high toxicity. However, it is still difficult to detect ultra-trace mercury species due to their biochemical transformation in complex samples. To establish a simpler and more sensitive method for pre-concentration and determination of trace mercury species, molybdenum disulfide (MoS2) nanosheets with sulfur-rich characteristics and enlarged interlayer spacing were prepared by a hydrothermal method coupled with a sonication-assisted liquid exfoliation method and acted as solid-phase extraction adsorbent. The nano-MoS2 had high adsorption capacity, fast adsorption rate and excellent selectivity towards mercury ions (Hg2+), methyl mercury (MeHg+) and ethyl mercury (EtHg+) in a wide pH range and complex matrices. And it could be easily regenerated by 4 mol L-1 HCl and reused several times. After optimizing HPLC-UV-HG-AFS conditions, a great linearity (1.0-10.0 µg L-1, R 2 = 0.999 for Hg2+, MeHg+ and EtHg+), lower detection limits (0.017, 0.037 and 0.021 ng mL-1 for Hg2+, MeHg+ and EtHg+, respectively), relative standard deviations (<5%) and addition recoveries of the samples within 82.75-113.38% were observed. In summary, trace inorganic and organic mercury species in environmental and biological samples could be selectively enriched by the prepared nano-MoS2 and efficiently seperated and detected by HPLC-UV-HG-AFS. The present study will help provide a better strategy for environmental monitoring and health assessment of mercury pollutants.