Optogenetic induction of caspase-8 mediated apoptosis by employing Arabidopsis cryptochrome 2.

Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.

CD69 serves as a potential diagnostic and prognostic biomarker for hepatocellular carcinoma.

The prevalence and mortality of hepatocellular carcinoma (HCC) are still increasing. This study aimed to identify potential therapeutic targets related to patient prognosis. Data were downloaded from TCGA, GSE25097, GSE36376, and GSE76427 datasets. Differential analysis and enrichment analysis were performed in HCC. Cell deaths were evaluated, and least absolute shrinkage and selection operator regression (LASSO) regression was analyzed to screen candidate genes. Additionally, immune cell infiltration in HCC was assessed. We identified 4088 common DEGs with the same direction of differential expression in all four datasets, they were mainly enriched in immunoinflammation and cell cycle pathways. Apoptosis was significantly suppressed in HCC in GSEA and GSVA. After LASSO regression analysis, we screened CD69, CDC25B, MGMT, TOP2A, and TXNIP as candidate genes. Among them, CD69 significantly influenced the overall survival of HCC patients in both TCGA and GSE76427. CD69 may be a protective factor for outcome of HCC patients. In addition, CD69 was positive correlation with T cells and CD3E. CD69, CDC25B, MGMT, TOP2A, and TXNIP were potential diagnostic and prognostic target for HCC, especially CD69.

A scoring model for diagnosis of tuberculous pleural effusion.

BACKGROUND: Due to the low efficiency of a single clinical feature or laboratory variable in the diagnosis of tuberculous pleural effusion (TBPE), the diagnosis of TBPE is still challenging. This study aimed to build a scoring diagnostic model based on laboratory variables and clinical features to differentiate TBPE from non-tuberculous pleural effusion (non-TBPE). METHODS: A retrospective study of 125 patients (63 with TBPE; 62 with non-TBPE) was undertaken. Univariate analysis was used to select the laboratory and clinical variables relevant to the model composition. The statistically different variables were selected to undergo binary logistic regression. Variables B coefficients were used to define a numerical score to calculate a scoring model. A receiver operating characteristic (ROC) curve was used to calculate the best cut-off value and evaluate the performance of the model. Finally, we add a validation cohort to verify the model. RESULTS: Six variables were selected in the scoring model: Age ≤ 46 years old (4.96 points), Male (2.44 points), No cancer (3.19 points), Positive T-cell Spot (T-SPOT) results (4.69 points), Adenosine Deaminase (ADA) ≥ 24.5U/L (2.48 point), C-reactive Protein (CRP) ≥ 52.8 mg/L (1.84 points). With a cut-off value of a total score of 11.038 points, the scoring model's sensitivity, specificity, and accuracy were 93.7%, 96.8%, and 99.2%, respectively. And the validation cohort confirms the model with the sensitivity, specificity, and accuracy of 92.9%, 93.3%, and 93.1%, respectively. CONCLUSION: The scoring model can be used in differentiating TBPE from non-TBPE.

Arabidopsis cryptochrome 2 forms photobodies with TCP22 under blue light and regulates the circadian clock.

Cryptochromes are blue light receptors that regulate plant growth and development. They also act as the core components of the central clock oscillator in animals. Although plant cryptochromes have been reported to regulate the circadian clock in blue light, how they do so is unclear. Here we show that Arabidopsis cryptochrome 2 (CRY2) forms photobodies with the TCP22 transcription factor in response to blue light in plant cells. We provide evidence that PPK kinases influence the characteristics of these photobodies and that together these components, along with LWD transcriptional regulators, can positively regulate the expression of CCA1 encoding a central component of the circadian oscillator.

Small RNA Sequencing Reveals Exosomal miRNAs as Unique Feature Markers in Unprovoked Venous Thromboembolism.

INTRODUCTION: Venous thromboembolism (VTE) is a common cardiovascular disease. MicroRNAs (miRNAs) play a key role in VTE; however, the role of exosomal miRNAs in VTE remains unknown. Therefore, we aimed to identify key exosomal miRNAs and their potential mechanisms in VTE. METHODS: We collected 31 samples from unprovoked VTE patients and 25 samples from healthy individuals. Exosomal miRNA sequencing was performed on 11 unprovoked VTE samples and 9 normal samples, and the remaining samples were used to verify the expression level of candidate 9 miRNAs in VTE and normal samples. The sequencing data were used to analyze exosomal miRNA expression. Meanwhile, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to determine the potential biological functions of differentially expressed miRNA target genes. RESULTS: A total of 32 differentially expressed miRNAs were identified by sequencing. Among the 32 miRNAs, 23 miRNAs were upregulated (72%), and 9 miRNAs were downregulated (28%). In addition, we found that the biological functions and metabolic pathways of the target genes were related to hemostatic factors involved in VTE, indicating the regulation of differentially expressed miRNAs. We identified key miRNAs by constructing a miRNA-messenger RNA regulatory network and found that the target genes were related to VTE. We finally determined that the abnormal expression of 9 miRNAs is closely related to VTE, and the expression level between VTE and normal was verified through other samples. CONCLUSION: Our study identified a set of key miRNAs for future research on the molecular mechanisms of VTE.

Arabidopsis CIB3 regulates photoperiodic flowering in an FKF1-dependent way.

Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) are blue light receptors mediating light regulation of growth and development, such as photoperiodic flowering. CRY2 interacts with a basic helix-loop-helix transcription factor CIB1 in response to blue light to activate the transcription of the flowering integrator gene FLOWERING LOCUS T (FT). CIB1, CIB2, CIB4, and CIB5 function redundantly to promote flowering in a CRY2-dependent way and form various heterodimers to bind to the noncanonical E-box sequence in the FT promoter. However, the function of CIB3 has not been described. We discovered that CIB3 promotes photoperiodic flowering independently of CRY2. Moreover, CIB3 does not interact with CRY2 but interacts with CIB1 and functions synergistically with CIB1 to promote the transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1-CIB3 interaction in response to blue light.

Reconstituting Arabidopsis CRY2 Signaling Pathway in Mammalian Cells Reveals Regulation of Transcription by Direct Binding of CRY2 to DNA.

In response to blue light, cryptochromes photoexcite and interact with signal partners to transduce signal almost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light.

Using HEK293T Expression System to Study Photoactive Plant Cryptochromes.

Cryptochromes are photolyase-like blue light receptors that are conserved in plants and animals. Although the light-dependent catalytic mechanism of photolyase is well studied, the photochemical mechanism of cryptochromes remains largely unknown. Lack of an appropriate protein expression system to obtain photochemically active cryptochrome holoproteins is a technical obstacle for the study of plant cryptochromes. We report here an easy-to-use method to express and study Arabidopsis cryptochrome in HEK293T cells. Our results indicate that Arabidopsis cryptochromes expressed in HEK293T are photochemically active. We envision a broad use of this method in the functional investigation of plant proteins, especially in the large-scale analyses of photochemical activities of cryptochromes such as blue light-dependent protein-protein interactions.