A label-free and rapid fluorometric strategy for microRNA detection using CRISPR-Cas12a coupled with copper nanoparticles.

CRISPR-Cas12a with robust trans-cleavage activity were employed to mitigate background fluorescence signal, achieving sensitive detection of miRNA-21. The activation of trans-cleavage activity of Cas12a was achieved by utilizing cDNA as a trigger. Upon the presence of target miRNA-21, cDNA hybridizes with it forming a DNA/RNA double-stranded structure. Exonuclease III (ExoIII) facilitates the degradation of cDNA, releasing the target for subsequent cycles. Due to cDNA degradation, the trans-cleavage activity of Cas12a remains unactivated and does not disrupt the synthesis template of copper nanoparticles. Addition of Cu2+ and AA leads to the formation of highly fluorescent copper nanoparticles. Conversely, in absence of miRNA-21, intact cDNA activates trans-cleavage activity of Cas12a, resulting in degradation of the synthesis template and failure in synthesizing fluorescent copper nanoparticles. This method exhibits excellent selectivity with a low limit of detection (LOD) at 5 pM. Furthermore, we successfully applied this approach to determine miRNA-21 in cell lysates and human serum samples, providing a new approach for sensitive determination of biomarkers in biochemical research and disease diagnosis.

Chemical Targeting and Manipulation of Type III Secretion in the Phytopathogen Xanthomonas campestris for Control of Disease.

Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris.IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.

RBCK1 overexpression is associated with immune cell infiltration and poor prognosis in hepatocellular carcinoma.

RBCK1 is an important E3 ubiquitin ligase, which plays an important role in many major diseases. However, the function and mechanism of RBCK1 in pan-cancer and its association with immune cell infiltration have not been reported. The purpose of this study is to find out the expression of RBCK1 in cancer, and to explore the relationship between RBCK1 and the prognosis of patients. Our results show that the expression of RBCK1 is up-regulated in a variety of malignant tumors, and is closely related to the prognosis of patients. Further studies have shown that RBCK1 regulates protein expression in the nucleus and plays an important role in ribosome and valine, leucine, and isoleucine degradation. Genetic variation analysis showed that RBCK1 was mainly involved in missense mutations in multiple tumors, and mutated patients showed poor prognoses. Further studies showed that RBCK1 may be interacted with proteins such as RNRPB, MCRS1, TRIB3, MKKS and ARPC3. Through protein interaction analysis, we found 43 proteins interacting with RBCK1 in liver cancer. We also analyzed immune cell infiltration and found that RBCK1 expression was positively correlated with T cells and macrophages, while it was negatively correlated with neutrophils, NK cells, and DCs in liver cancer. Finally, we confirmed experimentally that RBCK1 can significantly inhibit the apoptosis and invasion of HCC. Therefore, we speculate that RBCK1 plays an important regulatory role in the occurrence and development of HCC.

Icaritin greatly attenuates ß-amyloid-induced toxicity in vivo.

AIMS: The accumulation and deposition of ß-amyloid (Aß) has always been considered a major pathological feature of Alzheimer's disease (AD). The latest and mainstream amyloid cascade hypothesis indicates that all the main pathological changes in AD are attributed to the accumulation of soluble Aß. However, the exploration of therapeutic drugs for Aß toxicity has progressed slowly. This study aims to investigate the protective effects of Icaritin on the Aß-induced Drosophila AD model and its possible mechanism. METHODS: To identify the effects of Icaritin on AD, we constructed an excellent Drosophila AD model named Aßarc (arctic mutant Aß42) Drosophila. Climbing ability, flight ability, and longevity were used to evaluate the effects of Icaritin on AD phenotypes. Aßarc was determined by immunostaining and ELISA. To identify the effects of Icaritin on oxidative stress, we performed the detection of ROS, hydrogen peroxide, MDA, SOD, catalase, GST, and Caspase-3. To identify the effects of Icaritin on energy metabolism, we performed the detection of ATP and lactate. Transcriptome analysis and qRT-PCR verifications were used to detect the genes directly involved in oxidative stress and energy metabolism. Mitochondrial structure and function were detected by an electron microscopy assay, a mitochondrial membrane potential assay, and a mitochondrial respiration assay. RESULTS: We discovered that Icaritin almost completely rescues the climbing ability, flight ability, and longevity of Aßarc Drosophila. Aßarc was dramatically reduced by Icaritin treatment. We also found that Icaritin significantly reduces oxidative stress and greatly improves impaired energy metabolism. Importantly, transcriptome analysis and qRT-PCR verifications showed that many key genes, directly involved in oxidative stress and energy metabolism, are restored by Icaritin. Next, we found that Icaritin perfectly restores the integrity of mitochondrial structure and function damaged by Aßarc toxicity. CONCLUSION: This study suggested that Icaritin is a potential drug to deal with the toxicity of Aßarc, at least partially realized by restoring the mitochondria/oxidative stress/energy metabolism axis, and holds potential for translation to human AD.

Identification and validation of potential diagnostic signature and immune cell infiltration for NAFLD based on cuproptosis-related genes by bioinformatics analysis and machine learning.

Background and aims: Cuproptosis has been identified as a key player in the development of several diseases. In this study, we investigate the potential role of cuproptosis-related genes in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Method: The gene expression profiles of NAFLD were obtained from the Gene Expression Omnibus database. Differential expression of cuproptosis-related genes (CRGs) were determined between NAFLD and normal tissues. Protein-protein interaction, correlation, and function enrichment analyses were performed. Machine learning was used to identify hub genes. Immune infiltration was analyzed in both NAFLD patients and controls. Quantitative real-time PCR was employed to validate the expression of hub genes. Results: Four datasets containing 115 NAFLD and 106 control samples were included for bioinformatics analysis. Three hub CRGs (NFE2L2, DLD, and POLD1) were identified through the intersection of three machine learning algorithms. The receiver operating characteristic curve was plotted based on these three marker genes, and the area under the curve (AUC) value was 0.704. In the external GSE135251 dataset, the AUC value of the three key genes was as high as 0.970. Further nomogram, decision curve, calibration curve analyses also confirmed the diagnostic predictive efficacy. Gene set enrichment analysis and gene set variation analysis showed these three marker genes involved in multiple pathways that are related to the progression of NAFLD. CIBERSORT and single-sample gene set enrichment analysis indicated that their expression levels in macrophages, mast cells, NK cells, Treg cells, resting dendritic cells, and tumor-infiltrating lymphocytes were higher in NAFLD compared with control liver samples. The ceRNA network demonstrated a complex regulatory relationship between the three hub genes. The mRNA level of these hub genes were further confirmed in a mouse NAFLD liver samples. Conclusion: Our study comprehensively demonstrated the relationship between NAFLD and cuproptosis, developed a promising diagnostic model, and provided potential targets for NAFLD treatment and new insights for exploring the mechanism for NAFLD.

Vitamin K2 protects against Aß42-induced neurotoxicity by activating autophagy and improving mitochondrial function in Drosophila.

OBJECTIVE: Alzheimer disease is characterized by progressive decline in cognitive function due to neurodegeneration induced by accumulation of Aß and hyperphosphorylated tau protein. This study was conducted to explore the protective effect of vitamin K2 against Aß42-induced neurotoxicity. METHODS: Alzheimer disease transgenic Drosophila model used in this study was amyloid beta with the arctic mutation expressed in neurons. Alzheimer disease flies were treated with vitamin K2 for 28 days after eclosion. Aß42 level in brain was detected by ELISA. Autophagy-related genes and NDUFS3, the core subunit of mitochondrial complex I, were examined using real-Time PCR (RT-PCR) and western blot analysis. RESULTS: Vitamin K2 improved climbing ability (P = 0.0105), prolonged lifespan (P < 0.0001) and decreased Aß42 levels (P = 0.0267), upregulated the expression of LC3 and Beclin1(P = 0.0012 and P = 0.0175, respectively), increased the conversion of LC3I to LC3II (P = 0.0206) and decreased p62 level (P =0.0115) in Alzheimer disease flies. In addition, vitamin K2 upregulated the expression of NDUFS3 (P = 0.001) and increased ATP production (P = 0.0033) in Alzheimer disease flies. CONCLUSION: It seems that vitamin K2 protect against Aß42-induced neurotoxicity by activation of autophagy and rescue mitochondrial dysfunction, which suggests that it may be a potential valuable therapeutic approach for Alzheimer disease.

Overexpressed PERK suppresses the neurodegenerative phenotypes in PINK1B9 flies by enhancing mitochondrial function.

PTEN-induced putative kinase 1 (PINK1) mutation induces autosomal recessive Parkinson's Disease (PD), mitochondrial dysfunction is the central pathogenic process. However, more and more studies presented the bulk of the damage to neurons with mitochondrial dysfunction stems from the endoplasmic reticulum (ER) stress. In mitochondria damaged PINK1B9 fly model how protein kinase RNA-like ER kinase (PERK) arm of ER stress functions remains a mystery. Thus, we generated both PERK overexpressed (PEK OE) and down expressed (PEK RNAi) PINK1B9 flies and monitored their motor activity. We found PEK OE decreased the abnormal wing posture rate and rescued PINK1B9 flies' motor activity. Furthermore, we observed the increased number of dopaminergic neurons of protocerebral posterior lateral 1 (PPL1) and the tyrosine hydroxylase (TH) protein levels in PINK1B9 flies. When testing the mitochondrial morphology in flight muscle with TEM, we found that the shape of the mitochondria became normal. The ATP levels of flight muscle tissues were significantly elevated in PEK OE PINK1B9 flies with the increased function of mitochondrial Electron Transport Chain (ETC) Complex I (CI) but not Complex Ⅱ (CⅡ) which is further confirmed by oxygen consumption experiments, Western Blot, and RT-PCR to examine the corresponding subunits. We suggest that overexpression of PERK can rescue PINK1B9 PD flies' pathogenic phenotypes and it is linked with the improved mitochondrial function especially CI of ETC but not CⅡ. Our findings may pave a way for the target of the drug for alleviating the suffering of PINK1 mutant autosomal recessive PD patients.

Wnt2 overexpression protects against PINK1 mutant­induced mitochondrial dysfunction and oxidative stress.

The PTEN induced putative kinase 1 (PINK1) mutation is the second most common cause of autosomal recessive adolescent Parkinson's disease (PD). Furthermore, mitochondrial disorders and oxidative stress are important mechanisms in the pathogenesis of PD. Numerous members of the Wnt family have been found to be associated with neurodegenerative diseases. Therefore, the present study investigated the role of the Wnt2 gene in PINK1B9 transgenic flies, which is a PD model, and its underlying mechanism. It was identified that overexpression of Wnt2 reduced the abnormality rate of PD transgenic Drosophila and improved their flight ability, while other intervention groups had no significant effect. Furthermore, an increase in ATP concentration normalized mitochondrial morphology, and increased the mRNA expression levels of NADH­ubiquinone oxidoreductase chain 1 (ND1), ND42, ND75, succinate dehydrogenase complex subunits B, Cytochrome b and Cyclooxygenase 1, which are associated with Wnt2 overexpression. Moreover, overexpression of Wnt2 in PD transgenic Drosophila resulted in the downregulation of reactive oxygen species and malondialdehyde production, and increased manganese superoxide dismutase (MnSOD), while glutathione was not significantly affected. It was found that overexpression of Wnt2 did not alter the protein expression of ß­catenin in PINK1B9 transgenic Drosophila, but did increase the expression levels of PPARG coactivator 1α (PGC­1α) and forkhead box sub­group O (FOXO). Collectively, the present results indicated that the Wnt2 gene may have a protective effect on PD PINK1B9 transgenic Drosophila. Thus, it was speculated that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 overexpression may be related to the PGC­1α/FOXO/MnSOD signaling pathway in PINK1 mutant transgenic Drosophila.

Withaferin A alleviates traumatic brain injury induced secondary brain injury via suppressing apoptosis in endothelia cells and modulating activation in the microglia.

Traumatic brain injury (TBI) is a major public health concern with high rates of morbidity and mortality worldwide. Currently used medications, though effective, are also associated with several adverse effects. Development of effective neuroprotective agents with fewer side-effects would be of clinical value. Previous studies have shown that withaferin compounds have a potential neuroprotective effect in nervous system disorders. However, the effect of withaferin compounds, especially withaferin A (WFA), on traumatic brain injury is unclear. In the present study, both in vivo and in vitro models were used to assess whether WFA could exert a neuroprotective effect after TBI and were used to explore the associated mechanisms. The results showed that WFA significantly improved neurobehavioral function in a dose-dependent fashion and alleviated histological alteration of injury to tissues in TBI mice. In vitro models of TBI revealed that dose-dependent WFA treatment increased the viability of SH-SY5Y cells. In addition, WFA treatment could attenuate blood-brain barrier disruption and brain edema via suppressing apoptosis in endothelial cells. Furthermore, both our in vivo and in vitro results reveal that WFA treatment could significantly reduce levels of several neuroinflammation cytokines (IL-1ß, IL-6, and TNF-α), which correlate with an overall reduction in microglial activation. These data suggest that the neuroprotection by WFA is, at least in part, related to regulation of microglial activation and inhibition of vascular endothelial cell apoptosis. Taken together, these findings support further investigation of WFA as a promising therapeutic agent for promoting functional recovery after traumatic brain injury.