In-line RNA-based microreactor direct mass spectrometry for ultrasensitive and rapid assay of terminal deoxynucleotidyl transferase activity.

Terminal deoxynucleotidyl transferase (TdT), a unique template-independent DNA polymerase, plays a crucial role in the human adaptive immune system and is considered a promising biomarker for the diagnosis of various forms of acute or chronic leukemia. The accurate and sensitive detection of trace TdT is of pivotal importance to fulfill the significant medical interest in understanding its pathological functions and diagnosing TdT-related diseases. We hereby present an in-line RNA-based microreactor direct mass spectrometry (MS) method and its application for ultrasensitive, accurate, and rapid analysis of trace TdT activity in leukemic cell samples. A specially designed RNA-based microreactor is fabricated by immobilizing short RNA sequence via covalent Au-S bond on the inner surface of a capillary pre-modified with three-dimensional porous layer (PL) and Au nanoparticles (AuNPs). Utilizing this PL@Au@RNA microreactor, the signal of target TdT is conversed into reporter molecules (adenine), which exhibit a strong MS response. This conversion process enables efficient signal amplification and enhances detection sensitivity. The outlet end of the PL@Au@RNA microreactor is deliberately crafted into a porous tip, serving as an electrospray ionization (ESI) interface to directly couple to ESI-MS in-line. This design facilitates the direct transmission of the generated signaling molecules into the MS system, eliminating the need for laborious sample treatment procedures. By implementing this RNA-based microreactor in direct MS analysis, we have achieved remarkable sensitivity in detecting TdT activity with the limit-of-detection of 4 × 10-9 U, surpassing other reported methods in literature by three to four orders of magnitude. Furthermore, each assay requires a minimal sample volume of merely 10 nL. This method has successfully demonstrated its application in accurately and efficiently detecting TdT activity in leukemia cells, and its detection results are consistent with those obtained by ELISA kits.

CuO nanoparticles elicit intestinal immunotoxicity in zebrafish based on intestinal microbiota dysbiosis.

Copper II oxide nanoparticles (CuO NPs), a kind of widely used nanomaterial, have been detected in food and the environment, which has aroused widespread public concern. Recently, increasing data have suggested that intestinal microecology is closely related to immune homeostasis. However, the intestinal immunotoxicity induced by CuO NPs through intestinal microbiota is still unknown. Therefore, in this study, zebrafish were exposed to CuO NPs to explore intestinal immunotoxicity by evaluating physiological indicators, intestinal tissue injury, antioxidant enzyme activities, gene expression of immune factors, and changes in intestinal microbiota and its metabolites (short-chain fatty acids (SCFAs) and lipopolysaccharides (LPS)). The results revealed that the intestinal immunotoxicity of CuO NPs was mediated by the impact on intestinal microbiota and its metabolite levels. Specifically, changes were observed in the abundance of microbes that participated in the metabolism of SCFAs and LPS. The reduction in acetic acid, propionic acid and valeric acid upregulated GPR84 expression, and the decline in LPS levels further resulted in the suppression of the key immune regulatory pathways TLR4/MyD88/NF-κB, ultimately leading to intestinal immunotoxicity. This study would provide a scientific basis for the risk assessment of CuO NPs and a new perspective for research on the immunotoxicity of nanoparticles.

Point-of-care CRISPR/Cas biosensing technology: A promising tool for preventing the possible COVID-19 resurgence caused by contaminated cold-chain food and packaging.

The ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused great public health concern and has been a global threat due to its high transmissibility and morbidity. Although the SARS-CoV-2 transmission mainly relies on the person-to-person route through the respiratory droplets, the possible transmission through the contaminated cold-chain food and packaging to humans has raised widespread concerns. This review discussed the possibility of SARS-CoV-2 transmission via the contaminated cold-chain food and packaging by tracing the occurrence, the survival of SARS-CoV-2 in the contaminated cold-chain food and packaging, as well as the transmission and outbreaks related to the contaminated cold-chain food and packaging. Rapid, accurate, and reliable diagnostics of SARS-CoV-2 is of great importance for preventing and controlling the COVID-19 resurgence. Therefore, we summarized the recent advances on the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system-based biosensing technology that is promising and powerful for preventing the possible COVID-19 resurgence caused by the contaminated cold-chain food and packaging during the COVID-19 pandemic, including CRISPR/Cas system-based biosensors and their integration with portable devices (e.g., smartphone, lateral flow assays, microfluidic chips, and nanopores). Impressively, this review not only provided an insight on the possibility of SARS-CoV-2 transmission through the food supply chain, but also proposed the future opportunities and challenges on the development of CRISPR/Cas system-based detection methods for the diagnosis of SARS-CoV-2.

Metallothionein Attenuated Arsenic-Induced Cytotoxicity: The Underlying Mechanism Reflected by Metabolomics and Lipidomics.

Arsenic ions (As3+) have been recognized as a hazard that threatens the health of humans. Metallothionein (MT) rich in cysteine may provide favorable binding sites for chelation of As3+. However, the influence of MT on As3+-induced toxicity and the underlying mechanism are poorly understood, especially at the metabolic level. Herein, the effects of MT on As3+-induced toxicity were evaluated. Cell viability analysis suggested that MT alleviated As3+-induced cytotoxicity. The metabolic response of PC12 cells to As3+ investigated by lipidomics and metabolomics indicated that the presence of As3+ disrupted phospholipids metabolism and induced cell membrane damage. Moreover, energy and amino acid metabolism were perturbed by As3+. The perturbation of As3+ on metabolism was further illustrated by the decrease of the mitochondrial membrane potential and the rise of cellular reactive oxygen species (ROS). On the contrary, MT rescued As3+-induced metabolic disorder and suppressed ROS accumulation. In addition, the binding process between As3+ and MT was characterized. The results proved that the As3+-MT complex was formed and chelated As3+-scavenged ROS, thus alleviating the toxic effects of As3+. These results revealed that MT would be a potential agent to reduce As3+-induced cytotoxicity.

Salvia miltiorrhiza solution and its active compounds ameliorate human granulosa cell damage induced by H2O2.

The dried roots or rhizomes of Salvia miltiorrhiza Bge are commonly used in Chinese medicine to promote blood circulation and regulate menstruation. Salvianic acid A and salvianolic acid B are the main active water-soluble compounds in Salvia miltiorrhiza solution. The present study investigated the protective effect of Salvia miltiorrhiza solution and its active compounds in H2O2-induced cell damage of the human ovarian granulosa tumor cell line (KGN) in vitro, as well as its underlying mechanism. Cell viability was detected using a Cell Counting Kit-8 assay. In addition, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and tumor necrosis factor-α (TNF-α) were measured. Western blotting was performed to detect the protein expression of cleaved caspase-3 and caspase-9. Furthermore, immunocytochemistry was used to detect the expression of TNF-α. It was demonstrated that Salvia miltiorrhiza solution, salvianic acid A and salvianolic acid B did not affect the viability of KGN cells. Additionally, salvianic acid A and salvianolic acid B significantly reduced the H2O2-induced increased MDA levels, and reversed the H2O2-induced suppression of SOD and GSH activities in KGN cells (P<0.05). Treatment with Salvia miltiorrhiza solution, salvianic acid A and salvianolic acid B significantly reduced the overexpression of cleaved caspase-3, cleaved caspase-9 and TNF-α compared with the H2O2-treated group (P<0.05). Therefore, the present results indicated that Salvia miltiorrhiza solution and its main water-soluble compounds, salvianic acid A and salvianolic acid B, ameliorated KGN cell damage induced by H2O2.

Enhanced Cytotoxicity of Cadmium by a Sulfated Polysaccharide from Abalone.

Consumption of seafood is a common route of cadmium ion (Cd2+) exposure to consumers. The seafood matrices may alter the toxicity profile of Cd2+ due to the interaction between Cd2+ and biomacromolecules in seafood. In this study, enhanced cytotoxicity of Cd2+ was found in the presence of an abalone gonad sulfated polysaccharide (AGSP) and the mechanism was investigated at a metabolic level. The formation of the AGSP-Cd2+ complex was demonstrated by isothermal titration calorimetry. The level of reactive oxygen species (ROS) increased and mitochondrial membrane potential reduced upon exposure to the AGSP-Cd2+ complex as compared with those of Cd2+ exposure. The decreased cell viability after incubation with the AGSP-Cd2+ complex also suggested enhanced Cd2+ toxicity induced by AGSP. The metabolomics and lipidomics analysis revealed that, compared with the Cd2+ group, the AGSP-Cd2+ downregulated the phospholipid metabolism and resulted in more serious damage in the cellular membrane. The lipid metabolism disorder, in turn, amplified the generation of ROS, leading to a decrease in cell viability. These results provided new evidence of the enhanced Cd2+ toxicity upon interaction with seafood polysaccharides, and much attention should be paid to the effect of food ingredients on heavy metal ion toxicity.