Ratiometric and discriminative visualization of autophagic processes with a novel dual-responded lysosome-specific fluorescent probe.

BACKGROUND: Autophagy is a critical self-eating pathway involved in numerous physiological and pathological processes. Lysosomal degradation of dysfunctional organelles and invading microorganisms is central to the autophagy mechanism and essential for combating disease-related conditions. Therefore, monitoring fluctuations in the lysosomal microenvironment is vital for tracking the dynamic process of autophagy. Although much effort has been put into designing probes for measuring lysosomal viscosity or pH separately, there is a need to validate the concurrent imaging of the two elements to enhance the understanding of the dynamic progression of autophagy. METHODS: Probe HFI was synthesized in three steps and was developed to visualize changes in viscosity and pH within lysosomes for real-time autophagy tracking. Then, the spectrometric determination was carried out. Next, the probe was applied to image autophagy in cells under nutrient-deprivation or external stress. Additionally, the performance of HFI to monitor autophagy was employed to evaluate acetaminophen-induced liver injury. RESULTS: We constructed a ratiometric dual-responsive probe, HFI, with a large Stokes shift over 200 nm, dual-wavelength emission, and small background interference. The ratiometric fluorescent signal (R = I 610/I 460) of HFI had an excellent correlation with both viscosity and pH. More importantly, high viscosity and low pH had a synergistic promotion effect on the emission intensity of HFI, which enabled it to specially lit lysosomes without disturbing the inherent microenvironment. We then successfully used HFI to monitor intracellular autophagy induced by starvation or drugs in real-time. Interestingly, HFI also enabled us to visualize the occurrence of autophagy in the liver tissue of a DILI model, as well as the reversible effect of hepatoprotective drugs on this event. CONCLUSIONS: In this study, we developed the first ratiometric dual-responsive fluorescent probe, HFI, for real-time revealing autophagic details. It could image lysosomes with minimal perturbation to their inherent pH, allowing us to track changes in lysosomal viscosity and pH in living cells. Ultimately, HFI has great potential to serve as a useful indicator for autophagic changes in viscosity and pH in complex biological samples and can also be used to assess drug safety.

Establishing a Proteomics-Based Signature of AKR1C3-Related Genes for Predicting the Prognosis of Prostate Cancer.

Aldo-keto reductase family 1 member C3 (AKR1C3) plays an important role in prostate cancer (PCa) progression, particularly in castration-resistant prostate cancer (CRPC). It is necessary to establish a genetic signature associated with AKR1C3 that can be used to predict the prognosis of PCa patients and provide important information for clinical treatment decisions. AKR1C3-related genes were identified via label-free quantitative proteomics of the AKR1C3-overexpressing LNCaP cell line. A risk model was constructed through the analysis of clinical data, PPI, and Cox-selected risk genes. Cox regression analysis, Kaplan-Meier (K-M) curves, and receiver operating characteristic (ROC) curves were used to verify the accuracy of the model, and two external datasets were used to verify the reliability of the results. Subsequently, the tumor microenvironment and drug sensitivity were explored. Moreover, the roles of AKR1C3 in the progression of PCa were verified in LNCaP cells. MTT, colony formation, and EdU assays were conducted to explore cell proliferation and drug sensitivity to enzalutamide. Migration and invasion abilities were measured using wound-healing and transwell assays, and qPCR was used to assess the expression levels of AR target genes and EMT genes. CDC20, SRSF3, UQCRH, INCENP, TIMM10, TIMM13, POLR2L, and NDUFAB1 were identified as AKR1C3-associated risk genes. These risk genes, established using the prognostic model, can effectively predict the recurrence status, immune microenvironment, and drug sensitivity of PCa. Tumor-infiltrating lymphocytes and several immune checkpoints that promote cancer progression were higher in high-risk groups. Furthermore, there was a close correlation between the sensitivity of PCa patients to bicalutamide and docetaxel and the expression levels of the eight risk genes. Moreover, through in vitro experiments, Western blotting confirmed that AKR1C3 enhanced SRSF3, CDC20, and INCENP expression. We found that PCa cells with a high expression of AKR1C3 have high proliferation ability and high migration ability and were insensitive to enzalutamide. AKR1C3-associated genes had a significant role in the process of PCa, immune responses, and drug sensitivity and offer the potential for a novel model for prognostic prediction in PCa.

When type II diabetes mellitus meets COVID-19-Identification of the shared gene signatures and biological mechanism between the two diseases.

BACKGROUND: According to current studies, more than 20% of all patients diagnosed with COVID-19 globally have diabetes. Further, the mortality rate of these patients is 7.3%. Compared with non-diabetic COVID-19 patients, diabetic COVID-19 patients have higher rates of mortality and severe infection, suggesting that diabetes is associated with the severity of COVID-19 infection. This study aimed to analyse the relationship and susceptibility factors between COVID-19 and T2DM. METHODS: Using bioinformatics methods, potential targets for COVID-19 and T2DM were screened from GeneCards database. Potential targets of COVID-19 and T2DM were mapped to each other to identify overlapping targets, and a PPI network was constructed to extract the core target. The clusterProfiler package in R was used to analyse the function and pathway that core target involved. GO enrichment and KEGG pathway analysis were used to elucidate the correlation between COVID-19 and T2DM. RESULTS: A total of 277 potential pathogenic targets of COVID-19 were found, 282 potential targets were found for T2DM. Mapping of the potential COVID-19 and T2DM targets revealed 53 overlapping targets, with TNF as the core target. IL-17 signalling pathway was the most significant KEGG pathway involving TNF. CONCLUSIONS: The inflammatory cytokine, TNF, was identified as a core target between COVID-19 and T2DM, which induces inflammatory response mainly through the IL-17 signalling pathway, leading to aggravation of infection and increased difficulty in blood glucose control. This study provides a reference for further exploring the potential correlation and endogenous mechanisms between two seemingly independent and unrelated diseases-T2DM and COVID-19.

Photo-Activable Organosilver Nanosystem Facilitates Synergistic Cancer Theranostics.

Anticancer drug development is important for human health, yet it remains a tremendous challenge. Photodynamic therapy (PDT), which induces cancer cell apoptosis via light-triggered production of reactive oxygen species, is a promising method. However, it has minimal efficacy in subcellular targeting, hypoxic microenvironments, and deep-seated malignancies. Here, we constructed a breast cancer photo-activable theranostic nanosystem through the rational design of a synthetic lysosomal-targeted molecule with multifunctions as aggregation-induced near-infrared (NIR) emission, a photosensitizer (PDT), and organosilver (chemotherapy) for NIR imaging and synergistic cancer therapy. The synthetic molecule could self-assemble into nanoparticles (TPIMBS NPs) and be stabilized with amphiphilic block copolymers for enhanced accumulation in tumor sites through passive targeting while reducing the leakage in normal tissues. Through photochemical internalization, TPIMBS NPs preferentially concentrated in the lysosomes of cancer cells and generated reactive oxygen species (ROS) upon light irradiation, resulting in lysosomal rupture and release of PSs to the cytosol, which led to cell apoptosis. Further, the photoinduced release of Ag+ from TPIMBS NPs could act as chemotherapy, significantly improving the overall therapeutic efficacy by synergistic effects with PDT. This research sheds fresh light on the creation of effective cancer treatments.

Functional insights from targeted imaging BACE1: the first near-infrared fluorescent probe for Alzheimer's disease diagnosis.

BACKGROUND: ß-Secretase (BACE1) is the vital enzyme in the pathogenic processes of Alzheimer's disease (AD). However, the development of a powerful tool with sensitivity for BACE1 determination in vivo is a challenge. METHODS: A novel NIR fluorescent probe HBAE was synthetized from 2-hydroxy-3-methylbenzaldehyde and 2-amino-benzenethiol by 5 steps. The fluorescence mechanism in the ESIPT systems of HBAE probe was insighted with time-dependent density functional theory (TD-DFT) at the TDPBE0 level with the def2-TZVP approach. The corresponding docking between HBAE and BACE1 (PDB: 5I3Y) was performed through the ducking method by DOCK6.8. Then the BBB permeability of HBAE is verified by transwell orifice plate. 22-month-old male AD-model (5XFAD) mice and age-matched wild-type mice were employed to observe the brain kinetics by intravenous injection. Finally, Immunohistochemistry was performed on the AD brain section to reveal the levels of BACE1 in hippocampus and cortex areas and other regions in AD mice through the brain tissue slices by HBAE. RESULTS: The NIR fluorescent probe HBAE was successfully applied in imaging BACE1 in AD model mice. The capability of HBAE in reflecting different level of BACE1 was performed by the specific imaging of the hippocampus region. CONCLUSIONS: We reported the first ESIPT near-infrared fluorescence probe HBAE for monitoring endogenous BACE1 in the AD live model mice, thus offering a versatile chemical tool for visualizing in the pathological processes of AD live brains. Remarkably, high resolution images showed the localization of red fluorescence stains in hippocampus of the AD brain. This study provides a promising way for functional insights from protein BACE1 in vivo.

Environmentally Benign and User-Friendly In Situ Generation of Nitrile Imines from Hydrazones for 1,3-Dipolar Cycloaddition.

Nitrile imines are highly reactive and versatile dipoles and conventionally generated in situ from unstable hydrazonyl halides under basic conditions. Herein, we report the first green and user-friendly protocol for in situ generation of nitrile imines from Oxone-KBr oxidation of hydrazones and base-promoted dehydrobromination. The nitrile imines were demonstrated for 1,3-dipolar cycloaddition with various dipolarophiles, including alkene and alkyne groups. With its green nature, ease of operation, and air and moisture tolerance, we expect our method will find wide applications in organic synthesis.

NIR-I Dye-Based Probe: A New Window for Bimodal Tumor Theranostics.

Near-infrared (NIR, 650-1700 nm) bioimaging has emerged as a powerful strategy in tumor diagnosis. In particular, NIR-I fluorescence imaging (650-950 nm) has drawn more attention, benefiting from the high quantum yield and good biocompatibility. Since their biomedical applications are slightly limited by their relatively low penetration depth, NIR-I fluorescence imaging probes have been under extensive development in recent years. This review summarizes the particular application of the NIR-I fluorescent dye-contained bimodal probes, with emphasis on related nanoprobes. These probes have enabled us to overcome the drawbacks of individual imaging modalities as well as achieve synergistic imaging. Meanwhile, the application of these NIR-I fluorescence-based bimodal probes for cancer theranostics is highlighted.

siAKR1C3@PPA complex nucleic acid nanoparticles inhibit castration-resistant prostate cancer in vitro.

Introduction: AKR1C3, as a crucial androgenic enzyme, implicates the androgen biosynthesis and promoting prostate cancer cell growth in vitro. This study provides a new gene therapy strategy for targeting AKR1C3 to treat castration-resistant prostate cancer. Methods: siAKR1C3@PPA is assembled from PEG3500, PAMAM, Aptamer-PSMA, and siRNA for AKR1C3. We analyzed the relationship between AKR1C3 expression and the survival rate of prostate cancer patients based on the GEPIA online database to perform disease-free survival, and found that AKR1C3 may be an important factor leading to poor prognosis in prostate cancer. Considering AKR1C3 as a therapeutic target for castration-resistant prostate cancer, we constructed a complex nucleic acid nanoparticle, siAKR1C3@PPA to investigate the inhibitory effect on castration-resistant prostate cancer. Results: Aptamer-PSMA acts as a target to guide siAKR1C3@PPA into PSMA-positive prostate cancer cells and specifically down regulate AKR1C3. Cyclin D1 was decreased as a result of siAKR1C3@PPA treatment. Changes in Cyclin D1 were consistent with decreased expression of AKR1C3 in LNCaP-AKR1C3 cells and 22RV1 cells. Furthermore, in the LNCaP-AKR1C3 group, 1070 proteins were upregulated and 1015 proteins were downregulated compared to the LNCaP group according to quantitative 4D label-free proteomics. We found 42 proteins involved in cell cycle regulation. In a validated experiment, we demonstrated that PCNP and CINP were up-regulated, and TERF2 and TP53 were down-regulated by western blotting. Conclusion: We concluded that siAKR1C3@PPA may arrest the cell cycle and affect cell proliferation.

Evidence for a synergistic effect of post-translational modifications and genomic composition of eEF-1α on the adaptation of Phytophthora infestans.

Genetic variation plays a fundamental role in pathogen's adaptation to environmental stresses. Pathogens with low genetic variation tend to survive and proliferate more poorly due to their lack of genotypic/phenotypic polymorphisms in responding to fluctuating environments. Evolutionary theory hypothesizes that the adaptive disadvantage of genes with low genomic variation can be compensated for structural diversity of proteins through post-translation modification (PTM) but this theory is rarely tested experimentally and its implication to sustainable disease management is hardly discussed. In this study, we analyzed nucleotide characteristics of eukaryotic translation elongation factor-1α (eEF-lα) gene from 165 Phytophthora infestans isolates and the physical and chemical properties of its derived proteins. We found a low sequence variation of eEF-lα protein, possibly attributable to purifying selection and a lack of intra-genic recombination rather than reduced mutation. In the only two isoforms detected by the study, the major one accounted for >95% of the pathogen collection and displayed a significantly higher fitness than the minor one. High lysine representation enhances the opportunity of the eEF-1α protein to be methylated and the absence of disulfide bonds is consistent with the structural prediction showing that many disordered regions are existed in the protein. Methylation, structural disordering, and possibly other PTMs ensure the ability of the protein to modify its functions during biological, cellular and biochemical processes, and compensate for its adaptive disadvantage caused by sequence conservation. Our results indicate that PTMs may function synergistically with nucleotide codes to regulate the adaptive landscape of eEF-1α, possibly as well as other housekeeping genes, in P. infestans. Compensatory evolution between pre- and post-translational phase in eEF-1α could enable pathogens quickly adapting to disease management strategies while efficiently maintaining critical roles of the protein playing in biological, cellular, and biochemical activities. Implications of these results to sustainable plant disease management are discussed.

Construction and theoretical insights into the ESIPT fluorescent probe for imaging formaldehyde in vitro and in vivo.

We report the first ESIPT-based probe ABTB, for the highly sensitive and selective imaging of formaldehyde (FA). The various theoretical calculations have been systematically performed, and clearly unravel the lighting mechanism of the fluorescent probe for FA. Additionally, the probe was successfully applied in monitoring endogenous FA in the brain of AD mice.

Wash-free 3D imaging and detection of glioma with a novel neuropotential targeted AIE probe.

We herein developed a novel tetraarylimidazole-based AIE probe TPIG-NP to selectively image and quantitatively detect glioma. Due to the distinct negatively charged glioma cells, TPIG-NP with an opposite charge could achieve wash-free imaging of glioma cells and 3D multicellular spheroids.

Recent Advances in the Reactions of Cyclic Carbynes.

The acyclic organic alkynes and carbyne bonds exhibit linear shapes. Metallabenzynes and metallapentalynes are six- or five-membered metallacycles containing carbynes, whose carbine-carbon bond angles are less than 180°. Such distortion results in considerable ring strain, resulting in the unprecedented reactivity compared with acyclic carbynes. Meanwhile, the aromaticity of these metallacycles would stabilize the ring system. The fascinating combination of ring strain and aromaticity would lead to interesting reactivities. This mini review summarized recent findings on the reactivity of the metal-carbon triple bonds and the aromatic ring system. In the case of metallabenzynes, aromaticity would prevail over ring strain. The reactions are similar to those of organic aromatics, especially in electrophilic reactions. Meanwhile, fragmentation of metallacarbynes might be observed via migratory insertion if the aromaticity of metallacarbynes is strongly affected. In the case of metallapentalynes, the extremely small bond angle would result in high reactivity of the carbyne moiety, which would undergo typical reactions for organic alkynes, including interaction with coinage metal complexes, electrophilic reactions, nucleophilic reactions and cycloaddition reactions, whereas the strong aromaticity ensured the integrity of the bicyclic framework of metallapentalynes throughout all reported reaction conditions.