NOX4 promotes tumor progression through the MAPK-MEK1/2-ERK1/2 axis in colorectal cancer.

BACKGROUND: Metabolic reprogramming plays a key role in cancer progression and clinical outcomes; however, the patterns and primary regulators of metabolic reprogramming in colorectal cancer (CRC) are not well understood. AIM: To explore the role of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) in promoting progression of CRC. METHODS: We evaluated the expression and function of dysregulated and survival-related metabolic genes using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Consensus clustering was used to cluster CRC based on dysregulated metabolic genes. A prediction model was constructed based on survival-related metabolic genes. Sphere formation, migration, invasion, proliferation, apoptosis and clone formation was used to evaluate the biological function of NOX4 in CRC. mRNA sequencing was utilized to explore the alterations of gene expression NOX4 over-expression tumor cells. In vivo subcutaneous and lung metastasis mouse tumor model was used to explore the effect of NOX4 on tumor growth. RESULTS: We comprehensively analyzed 3341 metabolic genes in CRC and identified three clusters based on dysregulated metabolic genes. Among these genes, NOX4 was highly expressed in tumor tissues and correlated with worse survival. In vitro, NOX4 overexpression induced clone formation, migration, invasion, and stemness in CRC cells. Furthermore, RNA-sequencing analysis revealed that NOX4 overexpression activated the mitogen-activated protein kinase-MEK1/2-ERK1/2 signaling pathway. Trametinib, a MEK1/2 inhibitor, abolished the NOX4-mediated tumor progression. In vivo, NOX4 overexpression promoted subcutaneous tumor growth and lung metastasis, whereas trametinib treatment can reversed the metastasis. CONCLUSION: Our study comprehensively analyzed metabolic gene expression and highlighted the importance of NOX4 in promoting CRC metastasis, suggesting that trametinib could be a potential therapeutic drugs of CRC clinical therapy targeting NOX4.

Development of a clinical nomogram for prediction of response to neoadjuvant chemotherapy in patients with advanced gastric cancer.

BACKGROUND: The efficacy of neoadjuvant chemotherapy (NAC) in advanced gastric cancer (GC) is still a controversial issue. AIM: To find factors associated with chemosensitivity to NAC treatment and to provide the optimal therapeutic strategies for GC patients receiving NAC. METHODS: The clinical information was collected from 230 GC patients who received NAC treatment at the Central South University Xiangya School of Medicine Affiliated Haikou Hospital from January 2016 to December 2020. Least absolute shrinkage and selection operator logistic regression analysis was used to find the possible predictors. A nomogram model was employed to predict the response to NAC. RESULTS: In total 230 patients were finally included in this study, including 154 males (67.0%) and 76 females (33.0%). The mean age was (59.37 ± 10.60) years, ranging from 24 years to 80 years. According to the tumor regression grade standard, there were 95 cases in the obvious response group (grade 0 or grade 1) and 135 cases in the poor response group (grade 2 or grade 3). The obvious response rate was 41.3%. Least absolute shrinkage and selection operator analysis showed that four risk factors significantly related to the efficacy of NAC were tumor location (P < 0.001), histological differentiation (P = 0.001), clinical T stage (P = 0.008), and carbohydrate antigen 724 (P = 0.008). The C-index for the prediction nomogram was 0.806. The calibration curve revealed that the predicted value exhibited good agreement with the actual value. Decision curve analysis showed that the nomogram had a good value in clinical application. CONCLUSION: A nomogram combining tumor location, histological differentiation, clinical T stage, and carbohydrate antigen 724 showed satisfactory predictive power to the response of NAC and can be used by gastrointestinal surgeons to determine the optimal treatment strategies for advanced GC patients.

A novel cuproptosis-related signature predicts prognosis and immunotherapy efficacy in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the leading histological subtype of lung cancer worldwide, causing high annual mortality. Tsvetkov et al. recently found a new form of regulated cell death, termed cuproptosis. The prognostic value of cuproptosis-related gene signature in LUAD remains uncertain. METHODS: A training cohort is identified by the TCGA-LUAD dataset, whereas validation cohorts one and two are identified by GSE72094 and GSE68465, respectively. GeneCard and GSEA were used to extract genes related to cuproptosis. Cox regression, Kaplan-Meier regression, and LASSO regression were used to construct a gene signature. The model's applicability was evaluated by Kaplan-Meier estimators, Cox models, ROC, and tAUC across two independent validation cohorts. We examined the model's connections with other forms of regulated cell death. The immunotherapy ability of the signature was demonstrated by applying TMB, immune relevant signatures, and TIDE. The GSEA and immune infiltration analysis offer a better understanding of how the signature functions and the role of immune cells in its prognostic power. RESULTS: A ten-gene signature was built and demonstrated owning prognostic power by being applied to the validation cohorts. The GSEA uncovered that the unfolded protein response, glycolysis/gluconeogenesis, and MYC were highly related to the gene signature. The ten-gene signature is closely related to related genes of apoptosis, necroptosis, pyroptosis, and ferroptosis. Our signature may have utility in predicting immunotherapy efficacy in LUADs. Mast cells were identified as key players that support the predicting capacity of the ten-gene signature through the immune infiltrating analysis. CONCLUSIONS: The novel ten-gene signature associated with apoptosis in cuproptosis that we obtained may contribute to improved LUAD management strategies and the ability to predict response to LUAD immunotherapy. It is suggested that mast cell infiltration might be related to the prognostic power of this signature.

Diagnostic value of circular free DNA for colorectal cancer detection.

BACKGROUND: Minimally invasive or noninvasive, sensitive and accurate detection of colorectal cancer (CRC) is urgently needed in clinical practice. AIM: To identify a noninvasive, sensitive and accurate circular free DNA marker detected by digital polymerase chain reaction (dPCR) for the early diagnosis of clinical CRC. METHODS: A total of 195 healthy control (HC) individuals and 101 CRC patients (38 in the early CRC group and 63 in the advanced CRC group) were enrolled to establish the diagnostic model. In addition, 100 HC individuals and 62 patients with CRC (30 early CRC and 32 advanced CRC groups) were included separately to validate the model. CAMK1D was dPCR. Binary logistic regression analysis was used to establish a diagnostic model including CAMK1D and CEA. RESULTS: To differentiate between the 195 HCs and 101 CRC patients (38 early CRC and 63 advanced CRC patients), the common biomarkers CEA and CAMK1D were used alone or in combination to evaluate their diagnostic value. The area under the curves (AUCs) of CEA and CAMK1D were 0.773 (0.711, 0.834) and 0.935 (0.907, 0.964), respectively. When CEA and CAMK1D were analyzed together, the AUC was 0.964 (0.945, 0.982). In differentiating between the HC and early CRC groups, the AUC was 0.978 (0.960, 0.995), and the sensitivity and specificity were 88.90% and 90.80%, respectively. In differentiating between the HC and advanced CRC groups, the AUC was 0.956 (0.930, 0.981), and the sensitivity and specificity were 81.30% and 95.90%, respectively. After building the diagnostic model containing CEA and CAMK1D, the AUC of the CEA and CAMK1D joint model was 0.906 (0.858, 0.954) for the validation group. In differentiating between the HC and early CRC groups, the AUC was 0.909 (0.844, 0.973), and the sensitivity and specificity were 93.00% and 83.30%, respectively. In differentiating between the HC and advanced CRC groups, the AUC was 0.904 (0.849, 0.959), and the sensitivity and specificity were 93.00% and 75.00%, respectively. CONCLUSION: We built a diagnostic model including CEA and CAMK1D for differentiating between HC individuals and CRC patients. Compared with the common biomarker CEA alone, the diagnostic model exhibited significant improvement.

Conjugated structures based on quinazolinones and their application in fluorescent labeling.

As an alkaloid, quinazolinone exhibits excellent biological properties; structurally, it also has the potential to construct fluorescent probes with conjugated structures. In this work, probes 5a-c and 6b were obtained by introducing quinazolone into aldehydes with different numbers of double bonds. Their absorption maxima were located at 420-540 nm and their emission maxima were at 500-600 nm in solvents of different polarities. In particular, probe 5c showed significant fluorescence enhancement with the increase in viscosity due to the limited intramolecular rotation, and its fluorescence intensity in glycerol was 37.8 times higher than that in water. Moreover, probes 5a-c and 6b containing the NH structure showed sensitive response to pH, and their fluorescence intensity in alkaline solution (pH 9-11) was suddenly enhanced, which was elucidated with the help of theoretical calculation. In addition, the cell experiments showed that probes 5a and 5b had the ability to target mitochondria and probes 5c and 6b targeted lysosomes in HeLa cells. Furthermore, the viscosity-sensitive probe 5c could be used for monitoring changes in lysosomal viscosity in HeLa cells, which had important guiding significance for designing multi-response fluorogenic probes and promoting the advancement of cancer diagnosis.

Prospective association of family members' sugar-sweetened beverages intake with children's sugar-sweetened beverages consumption in China.

PURPOSE: We aimed to investigate whether parental and siblings' sugar-sweetened beverage (SSB) intake had prospective impact on children's SSB consumption, and the potential sex difference in these associations. METHODS: This study included a total of 904 children and their parents enrolled from 2004 to 2011 China Health and Nutrition Survey (CHNS) cohort study. SSB consumption information was estimated using a short dietary questionnaire and total energy intake was assessed with three-day 24-h dietary assessments at recruitment and follow-up surveys. Multivariate logistic or linear regression analyses were used to assess the association for SSB consumption between parents, siblings and children after adjusting for age, body mass index (BMI) z-score, household income and parental educational level. RESULTS: In this study, a majority (87.6%) of children consumed SSB. Among them, the median consumption of SSB was 70.3 ml/day per capita and 205.4 ml/day per consumer. Parental SSB consumption was relevant to children's SSB consumption, and this association was more pronounced in boys than in girls. Meanwhile, fathers seemed to have a stronger impact on whether children consume SSB than mothers which was reflected by lower P and higher OR. Additionally, children's SSB intake was prospectively associated with their older siblings' SSB consumption (P for trend < 0.03). CONCLUSIONS: Parental and older siblings' SSB consumption was relevant to children's SSB intake. Particularly, boys were more susceptible to parental impact than girls, and fathers seemed to have a greater influence on children than mothers.

Distinguishing cancer cells from normal cells with an organelle-targeted fluorescent marker.

In this paper we report a hemicyanine dye that is used to distinguish cancer cells from normal cells with its ability to target different organelles. Probe 1, a red emission hemicyanine functional dye, was connected to oxazolo[4,5-b]pyridine and diethylaminobenzene with a double bond. The maximum absorption peaks of probe 1 were located in the 509-552 nm range in organic solvents. Meanwhile, the probe possessed a high molar extinction coefficient (5.50 × 104 M-1 cm-1 in DMSO) with high photostability. The maximum emission wavelength of the probe ranged from 572 nm to 644 nm, and it also had a large Stokes shift (126 nm in DMSO). In particular, the probe showed weak fluorescence in water (Φ = 0.016), whereas it displayed strong fluorescence at 595 nm in ß-cyclodextrin (ß-CD) solution (Φ = 0.13). In addition, cell colocalization experiments showed that probe 1 (3 µM) was located in the endoplasmic reticulum in cancer cells, while it could target lysosomes in normal cells. What's more, further cell imaging experiments demonstrated that the average fluorescence intensity of probe 1 (0.3 µM) in cancer cells increased with the addition of ß-CD, but it did not occur in normal cells. The study provides a convenient way to distinguish cancer cells from normal ones, which has potential for application in the early detection of cancer.

Regorafenib combined with programmed cell death-1 inhibitor against refractory colorectal cancer and the platelet-to-lymphocyte ratio's prediction on effectiveness.

BACKGROUND: The effectiveness of regorafenib plus programmed cell death-1 (PD-1) inhibitor in treating microsatellite stable (MSS) metastatic colorectal cancer (mCRC) remains controversial. AIM: To investigate the benefits of regorafenib combined with PD-1 inhibitor in treating MSS mCRC and explore indicators predicting response. METHODS: This retrospective study included a total of 30 patients with microsatellite stable metastatic colorectal cancer treated with regorafenib combined with programmed cell death-1 inhibitor at Henan Provincial People's Hospital between December 2018 and December 2020. During a 4-wk treatment cycle, regorafenib was performed for 3 continuous weeks. PD-1 inhibitor was intravenously injected starting on the first day of the oral intake of regorafenib. We reviewed tumor response, progression-free survival (PFS), overall survival, and treatment-related adverse events (TRAEs) and evaluated association between platelet-to-lymphocyte ratio (PLR) and outcomes in this retrospective study. RESULTS: Stable disease and progressive disease were found in 18 (60.0%) and 12 (40.0%) patients, respectively. The disease control rate was 60.0%. The median follow-up time was 12.0 mo, and median PFS was 3.4 mo [95% confidence interval (CI): 2.2-4.6 mo]. Of the 12 patients with progressive disease, 10 (83.3%) had liver metastasis before starting the combined treatment. Among the 18 patients with SD, 10 (55.6%) did not have liver metastases. One patient without liver metastases at baseline was found with a substantially prolonged PFS of 11.2 mo. The liver metastasis, the choice of programmed cell death-1 inhibitor other than nivolumab or pembrolizumab and previous exposure to regorafenib was't associated with treatment outcome. The median PFS in the low-PLR group was 4.2 mo (95%CI: 3.5-4.9 mo), compared with 2.8 mo (95%CI: 1.4-4.2 mo) in the high-PLR group (P = 0.005). The major TRAEs included hand-foot syndrome (33.3%), hypertension (23.3%), malaise (20.0%), and gastrointestinal reaction (16.7%). The incidence of grade 3 TRAEs was 13.3% (4/30), which comprised abnormal capillary proliferation (n = 1), transaminase elevation (n = 1), and hand-foot syndrome (n = 2). No grade 4 or higher toxicity was observed. CONCLUSION: Regorafenib combined with PD-1 inhibitor could lead to a longer PFS in some patients with MSS mCRC. The PLR might be a prediction of the patient response to this therapy.

Toripalimab Combined With Hepatic Arterial Infusion Chemotherapy Versus Lenvatinib for Advanced Hepatocellular Carcinoma.

Purpose: Immunotherapy combined with chemotherapy have synergistic effects in multiple malignancies. We aimed to compare the efficacy and safety of toripalimab plus hepatic arterial infusion chemotherapy (HAIC) of oxaliplatin, fluorouracil, and leucovorin versus lenvatinib in advanced hepatocellular carcinoma (HCC). Materials and Methods: We conducted this retrospective study at 3 hospitals in China and eligible patients were 18 years or older and had a primary diagnosis of unresectable HCC with macroscopic vascular invasion and/or extrahepatic spread. These patients were treated with toripalimab plus HAIC or lenvatinib monotherapy. The primary endpoint was progression-free survival (PFS) and the secondary endpoints were overall survival (OS), disease control rate per response evaluation criteria in solid tumors (RECIST) 1.1, and objective response rate (ORR) per RECIST 1.1. The results were compared by Student's test or the chi-square test, and the survival curves were calculated by the Kaplan-Meier method, and propensity-score matching (PSM) was used to reduce bias. Results: A total of 118 patients were recruited for this study: 53 in the TorHAIC group and 65 in the lenvatinib group. We found that the TorHAIC group showed a longer PFS (9.3 [95% CI, 7.81-10.8] vs 4.8 months [95% CI, 3.31-6.29]; hazard ratio [HR] = 0.57, 95% CI, 0.38-0.85; p = .006), a longer OS (17.13 [95% CI, 13.99-20.27] vs 10.1 months [95% CI, 8.14-12.06]; HR = 0.5, 95% CI, 0.31 - 0.81; p = .005), a higher disease control rate (86.8% vs 69.2%, p = .002) and a higher ORR (47.2% vs 9.2%, p < .001) by RECIST criteria than the lenvatinib group. Both toripalimab plus HAIC and lenvatinib had acceptable safety profiles. No treatment-related deaths occurred in this study. In the propensity score-matched cohorts (47 pairs), the outcomes in the TorHAIC group were also better than those in the lenvatinib group (p < .05). Conclusion: Toripalimab plus HAIC was tolerable and effective in advanced HCC and the result needs to be confirmed in the phase III trial.

Construction of a single nucleotide variant score-related gene-based prognostic model in hepatocellular carcinoma: analysis of multi-independent databases and validation in vitro.

BACKGROUND: The accumulation of single nucleotide variants (SNVs) and the emergence of neoantigens can affect tumour proliferation and the immune microenvironment. However, the SNV-related immune microenvironment characteristics and key genes involved in hepatocellular carcinoma (HCC) are still unclear. We aimed to evaluate differences in the SNV-related immune microenvironment, construct a prognostic model and validate the key genes in vitro. METHODS: The categories of samples were defined by the expression of SNV score-related genes to evaluate the differences in mutational features, immune environment and prognosis. The survival model was constructed with survival-associated genes and verified in two independent test datasets. RCAN2, the key gene screened out for biofunction, was validated in vitro. RESULTS: IC2, among the three integrated clusters (IC1, IC2, IC3) classified by the 82 SNV score-related genes, was distinct from the rest in SNV score and immune cell infiltration, showing a better prognosis. Seven prognostic markers, HTRA3, GGT5, RCAN2, LGALS3, CXCL1, CLEC3B, and CTHRC1, were screened to construct a prognostic model. The survival model distinguished high-risk patients with poor prognoses in three independent datasets (log-rank P < 0.0001, 0.011, and 0.0068, respectively) with acceptable sensitivity and specificity. RCAN2 was inversely correlated with NK cell infiltration, and knockdown of RCAN2 promoted proliferation in HCC. CONCLUSIONS: This study revealed the characteristics of the HCC SNV-associated subgroup and screened seven latent markers for their accuracy of prognosis. Additionally, RCAN2 was preliminarily proven to influence proliferation in HCC and it had a close relationship with NK cell infiltration in vitro. With the capability to predict HCC outcomes, the model constructed with seven key differentially expressed genes offers new insights into individual therapy.

Viscosity sensitive endoplasmic reticulum fluorescent probes based on oxazolopyridinium.

A series of viscosity sensitive fluorescent probes 1a-e were synthesized by linking coumarin and oxazolopyridinium via dimethylene in this paper. The viscosity test of probes 1a-e indicated that the fluorescence intensity of the probes enhanced significantly with the increase of viscosity of the system (0.89-865 cP), and exhibited a nearly OFF-ON response to viscosity at 648 nm, 650 nm and 650 nm, respectively. In addition, cells still had a high survival rate after co-culturing with probes 1a-e for 12 h (94-98%). Meanwhile, the laser confocal experiment showed that the variation of the carbon chain length in the oxazolopyridinium could affect the subcellular region of the localization of the probes in cells. When the length of the carbon chain in oxazolopyridinium was between n-C7H15 and n-C12H23, probes 1b-d had the ability to target the endoplasmic reticulum in the cells. Moreover, probes 1b-d showed no significant change in fluorescence intensity after 35 min of continuous laser confocal irradiation, indicating that they had excellent anti-photobleaching properties.

Hepatic Arterial Infusion Chemotherapy of Oxaliplatin, Fluorouracil, and Leucovorin With or Without Sorafenib as Initial Treatment for Advanced Hepatocellular Carcinoma.

PURPOSE: Our previous study showed that hepatic arterial infusion chemotherapy (HAIC) using oxaliplatin, fluorouracil, and leucovorin (FOLFOX) plus sorafenib provided a significant survival benefit over sorafenib for advanced hepatocellular carcinoma. However, it is unclear whether the survival benefit should be attributed to the synergism between HAIC and sorafenib or just HAIC alone. We aim to compare HAIC using FOLFOX plus sorafenib with HAIC alone in patients with advanced hepatocellular carcinoma. MATERIALS AND METHODS: This was a retrospective study including 225 eligible patients treated with HAIC using FOLFOX (HAIC alone group, n=126, oxaliplatin 85 mg/m², leucovorin 400 mg/m², fluorouracil bolus 400 mg/m² and 2400 mg/m² for 46 hours, every 3 weeks) alone or HAIC plus sorafenib (soraHAIC group, n=99, sorafenib 400 mg twice daily). Survival curves were calculated by the Kaplan-Meier method, and propensity-score matching was used to reduce bias. RESULTS: The soraHAIC group showed a longer overall survival (12.9 [95% CI, 10.4-15.4] vs. 10.5 [95% CI, 9.5-11.5] months, HR=0.71 [95% CI, 0.53-0.96]; P=0.025), a better progression free survival (7.0 [95% CI, 5.3-8.8] vs. 5.3 [95% CI, 3.5-7.1] months, HR=0.76 [95% CI, 0.58-0.99]; P=0.046), and a higher disease control rate (RECIST 1.1: 74.8% vs. 61.1%, P=0.030) than the HAIC alone group. In multivariate analysis, soraHAIC was an independent favor factor for survival. In terms of the grade 3/4 adverse event, hand-foot skin reaction was more frequent in the soraHAIC group than the HAIC alone group. In the propensity-score matched cohorts (93 pairs), the overall survival, the progression free survival and disease control rates in the soraHAIC group were also better than those in the HAIC group (P<0.05). CONCLUSION: HAIC plus sorafenib may improve overall survival and progression free survival compared with HAIC alone as initial treatment for advanced hepatocellular carcinoma.

Acute myeloid leukemia with T lymphoblastic lymphoma: a case report and literature review.

Acute myeloid leukemia (AML) with T lymphoblastic lymphoma (T-LBL) is a hematologic tumor of two origins, myeloid and lymphoblastic, and is relatively rare in the same patient. We report a rare case of AML with T-LBL. After the patient was diagnosed, he received standard chemotherapy, which decreased the primitive bone marrow cell percentage from 84% to 5%; however, the enlarged superficial lymph nodes showed no obvious change in size. Immunohistochemistry revealed the following: cluster of differentiation (CD)3 (+), CD5 (+), CD7 (+), transmission disequilibrium test (TDT) (+), myeloperoxidase (MPO) (-), and lysozyme (Lys) (-). The lymph node morphology and immunohistochemical results indicated T-LBL. Therefore, the final diagnosis was AML with T-LBL, with both diseases occurring independently and concurrently.

Lenvatinib, toripalimab, plus hepatic arterial infusion chemotherapy versus lenvatinib alone for advanced hepatocellular carcinoma.

BACKGROUND: Lenvatinib is the first-line treatment for advanced hepatocellular carcinoma, but prognosis is still unsatisfactory. Recently, hepatic arterial infusion chemotherapy (HAIC), and immune checkpoint inhibitors showed promising results for advanced hepatocellular carcinoma. Considering different anti-malignancy mechanisms, combining these three treatments may improve outcomes. This study aimed to compare the efficacy and safety of lenvatinib, toripalimab, plus HAIC versus lenvatinib for advanced hepatocellular carcinoma. METHODS: This was a retrospective study including patients treated with lenvatinib [8 mg (⩽60 kg) or 12 mg (>60 kg) once daily] or lenvatinib, toripalimab plus HAIC [LeToHAIC group, lenvatinib 0-1 week prior to initial HAIC, 240 mg toripalimab 0-1 day prior to every HAIC cycle, and HAIC with FOLFOX regimen (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, 5-fluorouracil bolus 400 mg/m2 on day 1, and 5-fluorouracil infusion 2400 mg/m2 for 46 h, every 3 weeks)]. Progression-free survival, overall survival, objective response rate, and treatment-related adverse events were compared. RESULTS: From February 2019 to August 2019, 157 patients were included in this study: 71 in the LeToHAIC group and 86 in the lenvatinib group. The LeToHAIC group showed longer progression-free survival (11.1 versus 5.1 months, p < 0.001), longer overall survival (not reached versus 11 months, p < 0.001), and a higher objective response rate (RECIST: 59.2% versus 9.3%, p < 0.001; modified RECIST: 67.6% versus 16.3%, p < 0.001) than the lenvatinib group. In addition, 14.1% and 21.1% of patients in the LeToHAIC group achieved complete response of all lesions and complete response of the intrahepatic target lesions per modified RECIST criteria, respectively. Grade 3/4 treatment-related adverse events that were more frequent in the LeToHAIC group than in the lenvatinib group included neutropenia (8.5% versus 1.2%), thrombocytopenia (5.6% versus 0), and nausea (5.6% versus 0). CONCLUSIONS: Lenvatinib, toripalimab, plus HAIC had acceptable toxic effects and might improve survival compared with lenvatinib alone in advanced hepatocellular carcinoma.

The application of amide units in the construction of neutral functional dyes for mitochondrial staining.

To develop a new class of neutral fluorescent dyes with mitochondrial staining capacity, a series of functional dyes were obtained from Nile Red (2a-e) and coumarin (3a-e) with different amide compounds via Suzuki coupling reactions. The Nile Red derivatives (2a-e) emitted red fluorescence (590-660 nm) and coumarin derivatives (3a-e) showed blue emission (455-490 nm) in organic solvents. In addition, they exhibited high fluorescence quantum yields (0.27-0.98) in organic solvents and excellent photostability (>92%). Moreover, all of them possessed low cytotoxicity. More importantly, Nile Red borate (2) and coumarin borate (3) only accumulated in lipid droplets, while after being modified by different amide compounds, dyes 2a-e and 3a-e could successfully target mitochondria in HeLa cancer cells via confocal fluorescence experiments. This work provides a new strategy for the design of neutral cellular probes for mitochondrial staining.

The application of nitrogen heterocycles in mitochondrial-targeting fluorescent markers with neutral skeletons.

Four different neutral fluorescent markers containing nitrogen heterocycles (quinoxaline, 1H-pyrazolo[3,4-b]pyridine, 1H-indazole and 1H-pyrrolo[2,3-b]pyridine) as targeting groups were designed and prepared in order to screen out structural units for targeting mitochondria. Several classical fluorophores (coumarin, 1,8-naphthalimide and Nile Red) were connected with these heterocycles via Suzuki coupling reactions. The derivatives of coumarin (dyes 1a and 2a-c) and 1,8-naphthalimide (dyes 3a-c) fluoresced in the blue-green region, while the Nile Red derivatives (dyes 1b and 4a-c) fluoresced in the red light region. The optical properties of the classical fluorophores, such as emission properties and photostability, were retained in the new dyes. All of them showed low cytotoxicity. Confocal fluorescence experiments in L929 normal cells and HeLa cancer cells indicated that dyes 1a-b targeted dual sites of mitochondria and lipid droplets. Moreover, dyes 2a-c, 3a-c and 4a-c targeted mitochondria; meanwhile, there are only a few mitochondria-targeting markers with neutral skeletons. Furthermore, it was found that nitrogen heterocycles with N-H bonds can improve the mitochondrial targeting ability of partial neutral fluorophores.

A novel xanthylene-based effective mitochondria-targeting ratiometric cysteine probe and its bioimaging in living cells.

In this study, a mitochondria-specific fluorescent probe for efficient ratiometric detection of Cys was designed and investigated. Probe 1 is composed of a xanthylene skeleton and a benzyl group containing an acryloyl moiety. The probe showed excellent water solubility, good selectivity and sensitivity toward Cys over other analytes, and afforded an extremely low detection limit of 33.7 nM. The possible detection mechanism was ascertained by HRMS analysis. Moreover, probe 1 had excellent mitochondrial-targeting ability (the Pearson's correlation coefficient was 0.96), and was capable of monitoring endogenous Cys in living HeLa cells by dual channel ratiometric bioimaging, demonstrating its significant potential in biological applications.

Benzoxazine-based fluorescent probes with different auxochrome groups for cysteine detection.

Three 5H-benzo[a]phenoxazin-5-one-based (benzoresorufin and nile-red) Cysteine (Cys) detection probes have been comparatively designed and synthesized in this paper. The optical experiments exhibit probe 1b with a crotonoyl group has no response toward Cys; while probes 1a and 1c have the same reaction site (acryloyl group), their optical responses to Cys are quite different. The benzoresorufin-based-probe 1a shows a turn-on fluorescence response (118-fold) to Cys at 631 nm and affords a very low detection limit (DL = 19.8 nM). Compared with probe 1a, the nile-red-based probe 1c displays gradually diminishing fluorescence intensity with increased Cys concentration at 665 nm. And the notable different fluorescence response mechanisms of probes 1a and 1c toward Cys can be interpreted by HRMS and time-dependent density functional theorety (TDDFT) calculations. Furthermore, both of the two probes indicate high sensitivity and selectivity toward Cys over other similar structured amino acids including homocysteine (Hcy) and glutathione (GSH). Further cellular applications of the two probes have been successfully performed in HeLa cells.

Fluorescent hydrogen sulfide probes based on azonia-cyanine dyes and their imaging applications in organelles.

Three hydrogen sulfide (H2S) probes based on an azonia-cyanine skeleton were successfully designed and prepared. Probe 1a, containing 4-chloro-7-nitro-1,2,3-benzoxadiazole connected to the cyanine dye, had an emission at 660 nm that was enhanced 4.5-fold by the reduced photoinduced electron transfer process when reacting with H2S. Probes 1b and 1c were constructed from cyanine dyes with electron withdrawing 2,4-dinitrophenyl and 7-nitrobenzo[c] [1,2,5]oxadiazol-4-yl groups, respectively. Probes 1b and 1c gave off-on type responses with 169- and 17-fold fluorescent enhancements at 639 nm with H2S. Their emission properties were influenced by intramolecular hydrogen bonds and intramolecular charge transfer processes. The detection limits of probes 1a-1c were calculated at 178, 121, and 9.6 nM, respectively. The intracellular imaging experiments with HeLa cells indicated probe 1a was a mitochondria-targeting H2S probe, while probes 1b and 1c were lysosome-targeting H2S probes.

Mechanism of exosomal microRNA-224 in development of hepatocellular carcinoma and its diagnostic and prognostic value.

BACKGROUND: Exosomes contain proteins, lipids, and biological molecules such as DNA and RNA. Nucleic acids in exosomes are a group of molecules that can act as biomarkers. Currently, there are many reports on exosomal microRNAs, which are ideal biomarkers for the early diagnosis of cancer. However, there are few reports on the role of exosomal microRNAs in the diagnosis and prognosis of hepatocellular carcinoma (HCC). AIM: To understand the mechanism of exosomal microRNA-224 (miR-224) in the development of HCC and evaluate its diagnostic and prognostic value. METHODS: Cell culture and transfection of exosomal miRNA-224, real-time quantitative PCR, luciferase reporter assay, and other methods were used to find new biomarkers related to the development of HCC that can be used to diagnose HCC and predict HCC prognosis. RESULTS: By targeting glycine N-methyltransferase, incubating exosomes with miR-224 mimic resulted in a significant increase in cell proliferation compared to that of the control group, while incubation with the miR-224 inhibitor significantly reduced cell proliferation. The same results were obtained for the cell invasion assay. Serum exosomal miR-224 did have some ability to differentiate patients with HCC from healthy controls, with an area under the curve of 0.910, and HCC patients with higher serum exosomal miR-224 expression had lower overall survival. CONCLUSION: Exosomal miR-224 is a tumor promotor and can be a marker of diagnosis and prognosis of HCC patients, however, its ability to distinguish liver diseases needs further verification.