Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets.

Sensors designed based on the trans-cleavage activity of CRISPR/Cas12a systems have opened up a new era in the field of biosensing. The current design of CRISPR/Cas12-based sensors in the "on-off-on" mode mainly focuses on programming the activator strand (AS) to indirectly switch the trans-cleavage activity of Cas12a in response to target information. However, this design usually requires the help of additional auxiliary probes to keep the activator strand in an initially "blocked" state. The length design and dosage of the auxiliary probe need to be strictly optimized to ensure the lowest background and the best signal-to-noise ratio. This will inevitably increase the experiment complexity. To solve this problem, we propose using AS after the "RESET" effect to directly regulate the Cas12a enzymatic activity. Initially, the activator strand was rationally designed to be embedded in a hairpin structure to deprive its ability to activate the CRISPR/Cas12a system. When the target is present, target-mediated strand displacement causes the conformation change in the AS, the hairpin structure is opened, and the CRISPR/Cas12a system is reactivated; the switchable structure of AS can be used to regulate the degree of activation of Cas12a according to the target concentration. Due to the advantages of low background and stability, the CRISPR/Cas12a-based strategy can not only image endogenous biomarkers (miR-21) in living cells but also enable long-term and accurate imaging analysis of the process of exogenous virus invasion of cells. Release and replication of virus genome in host cells are indispensable hallmark events of cell infection by virus; sensitive monitoring of them is of great significance to revealing virus infection mechanism and defending against viral diseases.

Multifunctional DNA Nanoflower Applied for High Specific Photodynamic Cancer Therapy In Vivo.

Photodynamic therapy (PDT) is a newly emerged strategy for disease treatment. One challenge of the application of PDT drugs is the side-effect caused by the non-specificity of the photosensitive molecules. Most of the photosensitizers may invade not only the pathogenic cells but also the normal cells. In recent, people tried to use special cargoes to deliver the drugs into target cells. DNA nanoflowers (NFs) are a kind of newly-emerged nanomaterial which constructed through DNA rolling cycle amplification (RCA) reaction. It is reported that the DNA NFs were suitable materials which have been widely applied as nanocargos for drug delivery in cancer chemotherapeutic treatment. In this paper, we have introduced a new multifunctional DNA NF which could be prepared through an one-pot RCA reaction. This proposed DNA NF contained a versatile AS1411 G-quadruplex moiety, which plays key roles not only for specific recognition of cancer cells but also for near-infrared ray based photodynamic therapy when conjugating with a special porphyrin molecule. We demonstrated that the DNA NF showed good selectivity toward cancer cells, leading to highly efficient photo-induced cytotoxicity. Moreover, the in vivo experiment results suggested this DNA NF is a promising nanomaterial for clinical PDT.

Neuromodulatory effects of GnRH on the caudal neurosecretory Dahlgren cells in female olive flounder.

Gonadotropin-releasing hormone (GnRH) is considered a key player in reproduction. The caudal neurosecretory system (CNSS) is a unique neurosecretory structure of fish that may be involved in osmoregulation, nutrition, reproduction, and stress-related responses. However, a direct effect of GnRH on Dahlgren cells remains underexplored. Here, we examined the electrophysiological response of Dahlgren cell population of the CNSS to GnRH analog LHRH-A2 and the transcription of related key genes of CNSS. We found that GnRH increased overall firing frequency and may be changed the firing pattern from silent to burst or phasic firing in a subpopulation of Dahlgren cells. The effect of GnRH on a subpopulation of Dahlgren cells firing activity was blocked by the GnRH receptor (GnRH-R) antagonist cetrorelix. A positive correlation was observed between the UII and GnRH-R mRNA levels in CNSS or gonadosomatic index (GSI) during the breeding season. These findings are the first demonstration of the ability of GnRH acts as a modulator within the CNSS and add to our understanding of the physiological role of the CNSS in reproduction and seasonal adaptation.

Comparison of toxicity of Ti3 C2 and Nb2 C Mxene quantum dots (QDs) to human umbilical vein endothelial cells.

Recently, we developed highly fluorescent Ti3 C2 and Nb2 C Mxene quantum dots (QDs) for labeling of in vitro models. However, the mechanism of the toxicity of the prepared QDs was not explored before. In this study, we addressed the possible mechanism associated with cytotoxicity of the QDs to human umbilical vein endothelial cells (HUVECs). Exposure to up to 100 µg/ml Ti3 C2 but not Nb2 C QDs for 24 h significantly induced cytotoxicity. The exposure also increased intracellular Ti and Nb elements, indicating the internalization of both types of QDs. None of the QDs promoted interleukin 6 (IL-6) and IL-8 releases. Rather, Ti3 C2 QDs significantly reduced IL-6 and IL-8 release, indicating that the toxicity of Ti3 C2 QDs was not due to elevated inflammatory responses. Exposure to Ti3 C2 but not Nb2 C QDs resulted in increased LC3B-II/I ratio and beclin-1 proteins, biomarkers of autophagy, as well as the accumulation of autophagic substance p62. Ti3 C2 QDs also more effectively promoted pro-caspase 3 but not pro-caspase 8 compared with Nb2 C QDs. Furthermore, pre-treatment with autophagic modulators altered the cytotoxicity of Ti3 C2 QDs, which further confirmed the role of autophagic dysfunction in Ti3 C2 QD-induced toxicity to HUVECs. In conclusion, the results from this study suggested that high levels of Ti3 C2 QDs could induce cytotoxicity to HUVECs by inducing the dysfunction of autophagy. Nb2 C QDs appeared to be more biocompatible to HUVECs compared with Ti3 C2 QDs at the same mass concentrations, which suggested a role of composition of Mxene QDs to determine their toxicity to human endothelial cells.

Epidemiology and prognostic nomogram for locally advanced gastric signet ring cell carcinoma: A population-based study.

BACKGROUND: Gastric signet ring cell carcinoma (GSRC) represents a specific subtype of gastric cancer renowned for its contentious epidemiological features, treatment principles, and prognostic factors. AIM: To investigate the epidemiology of GSRC and establish an improved model for predicting the prognosis of patients with locally advanced GSRC (LAGSRC) after surgery. METHODS: The annual rates of GSRC incidence and mortality, covering the years 1975 to 2019, were extracted from the Surveillance, Epidemiology, and End Results (SEER) database to explore the temporal trends in both disease incidence and mortality rates using Joinpoint software. The clinical data of 3793 postoperative LAGSRC patients were collected from the SEER database for the analysis of survival rates. The Cox regression model was used to explore the independent prognostic factors for overall survival (OS). The risk factors extracted were used to establish a prognostic nomogram. RESULTS: The overall incidence of GSRC increased dramatically between 1975 and 1998, followed by a significant downward trend in incidence after 1998. In recent years, there has been a similarly optimistic trend in GSRC mortality rates. The trend in GSRC showed discrepancies based on age and sex. Receiver operating characteristic curves, calibration curves, and decision curve analysis for 1-year, 3-year, and 5-year OS demonstrated the high discriminative ability and clinical utility of this nomogram. The area under the curve indicated that the performance of the new model outperformed that of the pathological staging system. CONCLUSION: The model we established can aid clinicians in the early prognostication of LAGSRC patients, resulting in improved clinical outcomes by modifying management strategies and patient health care.

Recent Development of Advanced Fluorescent Molecular Probes for Organelle-Targeted Cell Imaging.

Fluorescent molecular probes are very powerful tools that have been generally applied in cell imaging in the research fields of biology, pathology, pharmacology, biochemistry, and medical science. In the last couple of decades, numerous molecular probes endowed with high specificity to particular organelles have been designed to illustrate intracellular images in more detail at the subcellular level. Nowadays, the development of cell biology has enabled the investigation process to go deeply into cells, even at the molecular level. Therefore, probes that can sketch a particular organelle's location while responding to certain parameters to evaluate intracellular bioprocesses are under urgent demand. It is significant to understand the basic ideas of organelle properties, as well as the vital substances related to each unique organelle, for the design of probes with high specificity and efficiency. In this review, we summarize representative multifunctional fluorescent molecular probes developed in the last decade. We focus on probes that can specially target nuclei, mitochondria, endoplasmic reticulums, and lysosomes. In each section, we first briefly introduce the significance and properties of different organelles. We then discuss how probes are designed to make them highly organelle-specific. Finally, we also consider how probes are constructed to endow them with additional functions to recognize particular physical/chemical signals of targeted organelles. Moreover, a perspective on the challenges in future applications of highly specific molecular probes in cell imaging is also proposed. We hope that this review can provide researchers with additional conceptual information about developing probes for cell imaging, assisting scientists interested in molecular biology, cell biology, and biochemistry to accelerate their scientific studies.