Precise Detection of Viral RNA by Programming Multiplex Rolling Circle Amplification and Strand Displacement.

Detection of viral infections (e.g., SARS-CoV-2) with high precision is critical to disease control and treatment. There is an urgent need to develop point-of-care detection methods to complement the gold standard laboratory-based PCR assay with comparable sensitivity and specificity. Herein, we developed a method termed mCAD to achieve ultraspecific point-of-care detection of SARS-CoV-2 RNA while maintaining high sensitivity by programming multiplex rolling circle amplification and toehold-mediated strand displacement reactions. RCA offers sufficient amplification of RNA targets for subsequent detection. Most importantly, a multilayer of detection specificity is implemented into mCAD via sequence-specific hybridization of nucleic acids across serial steps of this protocol to fully eliminate potential false-positive detections. Using mCAD, we demonstrated a highly specific, sensitive, and convenient visual detection of SARS-CoV-2 RNA from both synthetic and clinical samples, exhibiting performance comparable to qPCR. We envision that mCAD will find its broad applications in clinical prospects for nucleic acid detections readily beyond SARS-CoV-2 RNA.

Construction of a risk stratification model integrating ctDNA to predict response and survival in neoadjuvant-treated breast cancer.

BACKGROUND: The pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) of breast cancer is closely related to a better prognosis. However, there are no reliable indicators to accurately identify which patients will achieve pCR before surgery, and a model for predicting pCR to NAC is required. METHODS: A total of 269 breast cancer patients in Shandong Cancer Hospital and Liaocheng People's Hospital receiving anthracycline and taxane-based NAC were prospectively enrolled. Expression profiling using a 457 cancer-related gene sequencing panel (DNA sequencing) covering genes recurrently mutated in breast cancer was carried out on 243 formalin-fixed paraffin-embedded tumor biopsies samples before NAC from 243 patients. The unique personalized panel of nine individual somatic mutation genes from the constructed model was used to detect and analyze ctDNA on 216 blood samples. Blood samples were collected at indicated time points including before chemotherapy initiation, after the 1st NAC and before the 2nd NAC cycle, during intermediate evaluation, and prior to surgery. In this study, we characterized the value of gene profile mutation and circulating tumor DNA (ctDNA) in combination with clinical characteristics in the prediction of pCR before surgery and investigated the prognostic prediction. The median follow-up time for survival analysis was 898 days. RESULTS: Firstly, we constructed a predictive NAC response model including five single nucleotide variant (SNV) mutations (TP53, SETBP1, PIK3CA, NOTCH4 and MSH2) and four copy number variation (CNV) mutations (FOXP1-gain, EGFR-gain, IL7R-gain, and NFKB1A-gain) in the breast tumor, combined with three clinical factors (luminal A, Her2 and Ki67 status). The tumor prediction model showed good discrimination of chemotherapy sensitivity for pCR and non-pCR with an AUC of 0.871 (95% CI, 0.797-0.927) in the training set, 0.771 (95% CI, 0.649-0.883) in the test set, and 0.726 (95% CI, 0.556-0.865) in an extra test set. This tumor prediction model can also effectively predict the prognosis of disease-free survival (DFS) with an AUC of 0.749 at 1 year and 0.830 at 3 years. We further screened the genes from the tumor prediction model to establish a unique personalized panel consisting of 9 individual somatic mutation genes to detect and analyze ctDNA. It was found that ctDNA positivity decreased with the passage of time during NAC, and ctDNA status can predict NAC response and metastasis recurrence. Finally, we constructed the chemotherapy prediction model combined with the tumor prediction model and pretreatment ctDNA levels, which has a better prediction effect of pCR with the AUC value of 0.961. CONCLUSIONS: In this study, we established a chemotherapy predictive model with a non-invasive tool that is built based on genomic features, ctDNA status, as well as clinical characteristics for predicting pCR to recognize the responders and non-responders to NAC, and also predicting prognosis for DFS in breast cancer. Adding pretreatment ctDNA levels to a model containing gene profile mutation and clinical characteristics significantly improves stratification over the clinical variables alone.

Imaging the metabolic reprograming of fatty acid synthesis pathway enables new diagnostic and therapeutic opportunity for breast cancer.

BACKGROUND: Reprogrammed metabolic network is a key hallmark of cancer. Profiling cancer metabolic alterations with spatial signatures not only provides clues for understanding cancer biochemical heterogeneity, but also helps to decipher the possible roles of metabolic reprogramming in cancer development. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was used to characterize the expressions of fatty acids in breast cancer tissues. Specific immunofluorescence staining was further carried out to investigate the expressions of fatty acid synthesis-related enzymes. RESULTS: The distributions of 23 fatty acids in breast cancer tissues have been mapped, and the levels of most fatty acids in cancer tissues are significantly higher than those in adjacent normal tissues. Two metabolic enzymes, fatty acid synthase (FASN) and acetyl CoA carboxylase (ACC), which being involved in the de novo synthesis of fatty acid were found to be up-regulated in breast cancer. Targeting the up-regulation of FASN and ACC is an effective approach to limiting the growth, proliferation, and metastasis of breast cancer cells. CONCLUSIONS: These spatially resolved findings enhance our understanding of cancer metabolic reprogramming and give an insight into the exploration of metabolic vulnerabilities for better cancer treatment.

Inhibition of NADPH oxidase 4 attenuates lymphangiogenesis and tumor metastasis in breast cancer.

Lymphangiogenesis is thought to contribute to promote tumor cells to enter lymphatic vessels and plant at a secondary site. Endothelial cells are the cornerstone of the generation of new lymphatic vessels. NADPH oxidase 4 (Nox4) is the most abundant one of NADPH oxidases in endothelial cells and the most studied one in relevance with cancer. Our purpose is to analyze the relationship between Nox4 and lymphangiogenesis and find out whether the newborn lymphatic vessels lead to cancer metastasis. We first explored the expression of Nox4 in lymphatic endothelial cells of primary invasive breast tumors and human normal mammary glands using GEO databases and found that Nox4 was upregulated in primary invasive breast tumors samples. In addition, its high expression correlated with lymph node metastasis in breast cancer patients. Nox4 could increase the tube formation and lymphatic vessel sprouting in a three-dimensional setting. In vivo, inhibition of Nox4 in 4T1 tumor-bearing mice could significantly decrease the tumor lymphangiogenesis and metastasis. Nox4 may increase tumor lymphangiogenesis via ROS/ERK/CCL21 pathway and attract CCR7-positive breast cancer cells to entry lymphatic vessels and distant organs. In conclusion, our results show that Nox4 is a factor that promotes lymphangiogenesis and is a potential target of antitumor metastasis.

The choice of tissue fixative is a key determinant for mass spectrometry imaging based tumor metabolic reprogramming characterization.

The application of mass spectrometry imaging (MSI) for the study of spatiotemporal alterations of the metabolites in tumors has brought a number of significant biological results. At present, metabolite profiling based on MSI is typically performed on frozen tissue sections; however, the majority of clinical specimens need to be fixed in tissue fixative to avoid autolysis and to preserve antigenicity. In this study, we present the global impacts of different fixatives on the MS imaging of gastric cancer tissue metabolites. The MSI performances of 17 kinds of metabolites, such as amino acids, polyamines, cholines, organic acids, polypeptides, nucleotides, nucleosides, nitrogen bases, cholesterols, fatty acids, and phospholipids, in untreated, 10% formalin-, 4% paraformaldehyde-, acetone-, and 95% ethanol-fixed gastric cancer tissues were thoroughly explored for the first time. Furthermore, we also investigated the spatial expressions of 6 metabolic enzymes, namely, GLS, FASN, CHKA, PLD2, cPLA2, and EGFR, closely related to tumor-associated metabolites. Immunohistochemical staining carried out on the same tissue sections' which have undergone MSI analysis' suggests that enzymatic characterization is feasible after metabolite imaging. Combining the spatial signatures of metabolites and pathway-related metabolic enzymes in heterogeneous tumor tissues offers an insight to understand the complex tumor metabolism. Graphical abstract.

P-coumaric Acid: Advances in Pharmacological Research Based on Oxidative Stress.

P-coumaric acid is an important phenolic compound that is mainly found in fruits, vegetables, grains, and fungi and is also abundant in Chinese herbal medicines. In this review, the pharmacological research progress of p-coumaric acid in recent years was reviewed, with emphasis on its role and mechanism in oxidative stress-related diseases, such as inflammation, cardiovascular diseases, diabetes, and nervous system diseases. Studies have shown that p-coumaric acid has a positive effect on the prevention and treatment of these diseases by inhibiting oxidative stress. In addition, p-coumaric acid also has anti-tumor, antibacterial, anti-aging skin and other pharmacological effects. This review will provide reference and inspiration for further research on the pharmacological effects of p-coumaric acid.

Transcriptomic profiling of the thermal tolerance in two subspecies of the bay scallop Argopecten irradians.

The bay scallop is a eurythermal species with high economic value and now represents the most cultured bivalve species in China. Two subspecies of the bay scallop, the northern subspecies Argopecten irradians irradians Korean population (KK) and the southern subspecies Argopecten irradians concentricus (MM), exhibited distinct adaptations to heat stress. However, the molecular mechanism of heat resistance of the two subspecies remains unclear. In this study, we compared the transcriptomic responses of the two subspecies to heat stress and identified the involved differentially expressed genes (DEGs) and pathways. More DEGs were found in the KK than in the MM when exposed to high temperatures, indicating elevated sensitivity to thermal stress in the KK. Enrichment analysis suggests that KK scallops may respond to heat stress more swiftly by regulating GTPase activity. Meanwhile, MM scallops exhibited higher resistance to heat stress mainly by effective activation of their antioxidant system. Chaperone proteins may play different roles in responses to heat stress in the two subspecies. In both subspecies, the expression levels of antioxidants such as GST were significantly increased; the glycolysis process regulated by PC and PCK1 was greatly intensified; and both apoptotic and anti-apoptotic systems were significantly activated. The pathways related to protein translation and hydrolysis, oxidoreductase activity, organic acid metabolism, and cell apoptosis may also play pivotal roles in the responses to heat stress. The results of this study may provide a theoretical basis for marker-assisted breeding of heat-resistant strains.

Whole-Genome Re-sequencing and Transcriptome Reveal Candidate Genes and Pathways Associated with Hybrid Sterility in Hermaphroditic Argopecten Scallops.

The interspecific hybrid scallops generated from the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) showed significant heterosis in growth. However, its sterility limits large-scale hybridization and hinders the development of the scallop breeding industry. Hybrid sterility is regulated by plenty of genes and involves a range of biochemical and physiological transformations. In this study, whole-genome re-sequencing and transcriptomic analysis were performed in sterile and fertile hybrid scallops. The potential genetic variations and abnormally expressed genes were detected to explore the mechanism underlying hybrid sterility in hermaphroditic Argopecten scallops. Compared with fertile hybrids, 24 differentially expressed genes (DEGs) with 246 variations were identified to be related to fertility regulation, which were mainly enriched in germarium-derived egg chamber formation, spermatogenesis, spermatid development, mismatch repair, mitotic and meiotic cell cycles, Wnt signaling pathway, MAPK signaling pathway, calcium modulating pathway, and notch signaling pathway. Specifically, variation and abnormal expression of these genes might inhibit the progress of mitosis and meiosis, promote cell apoptosis, and impede the genesis and maturation of gametes in sterile hybrid scallops. Eleven DEGs (XIAP, KAZN, CDC42, MEIS1, SETD1B, NOTCH2, TRPV5, M- EXO1, GGT1, SBDS, and TBCEL) were confirmed by qRT-PCR validation. Our findings may enrich the determination mechanism of hybrid sterility and provide new insights into the use of interspecific hybrids for extensive breeding.

Detection of SARS-CoV-2 RNA with a plasmonic chiral biosensor.

The detection of SARS-CoV-2 infection is crucial for effective prevention and surveillance of COVID-19. In this study, we report the development of a novel detection assay named CENSOR that enables sensitive and specific detection of SARS-CoV-2 RNA using a plasmonic chiral biosensor in combination with CRISPR-Cas13a. The chiral biosensor was designed by assembling gold nanorods (AuNR) into three-dimensional plasmonic architectures of controllable chirality on a DNA origami template. This modular assembly mode enhances the flexibility and adaptability of the sensor, thereby improving its universality as a sensing platform. In the presence of SARS-CoV-2 RNA, the CRISPR-Cas13a enzyme triggers collateral cleavage of RNA molecules, resulting in a differential chiral signal readout by the biosensor compared to when there are no RNA targets present. Notably, even subtle variations in the concentration of SARS-CoV-2 RNA can provoke significant changes in chiral signals after preamplification of RNA targets (calculated LOD: 0.133 aM), which establishes the foundation for quantitative detection. Furthermore, CENSOR demonstrated high sensitivity and accuracy in detecting SARS-CoV-2 RNA from clinical samples, suggesting its potential application in clinical settings for viral detection beyond SARS-CoV-2.

Cyperotundone combined with adriamycin induces apoptosis in MCF-7 and MCF-7/ADR cancer cells by ROS generation and NRF2/ARE signaling pathway.

Breast cancer has become the most prevalent cancer, globally. Adriamycin is a first-line chemotherapeutic agent, however, cancer cells acquire resistance to it, which is one of the most common causes of treatment failure. ROS and NRF2 are essential oxidative stress factors that play a key role in the oxidative stress process and are associated with cancer. Our goal is to create novel therapeutic drugs or chemical sensitizers that will improve chemotherapy sensitivity. The optimal concentration and duration for MCF-7 and MCF-7/ADR cells in ADR and CYT were determined using the CCK-8 assay. We found that ADR + CYT inhibited the activity of MCF-7 and MCF-7/ADR cells in breast cancer, as well as causing apoptosis in MCF-7 and MCF-7/ADR cells and blocking the cell cycle in the G0/G1 phase. ADR + CYT induces apoptosis in MCF-7 and MCF-7/ADR cells through ROS generation and the P62/NRF2/HO-1 signaling pathway. In breast cancer-bearing nude mice, ADR + CYT effectively suppressed tumor development in vivo. Overall, our findings showed that CYT in combination with ADR has potent anti-breast cancer cell activity both in vivo and in vitro, suggesting CYT as the main drug used to improve chemosensitivity.