Ferroptosis and cuproptosis prognostic signature for prediction of prognosis, immunotherapy and drug sensitivity in hepatocellular carcinoma: development and validation based on TCGA and ICGC databases.

Background: Hepatocellular carcinoma (HCC) is a common malignancy. Ferroptosis and cuproptosis promote HCC spread and proliferation. While fewer studies have combined ferroptosis and cuproptosis to construct prognostic signature of HCC. This work attempts to establish a novel scoring system for predicting HCC prognosis, immunotherapy, and medication sensitivity based on ferroptosis-related genes (FRGs) and cuproptosis-related genes (CRGs). Methods: FerrDb and previous literature were used to identify FRGs. CRGs came from original research. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases included the HCC transcriptional profile and clinical information [survival time, survival status, age, gender, Tumor Node Metastasis (TNM) stage, etc.]. Correlation, Cox, and least absolute shrinkage and selection operator (LASSO) regression analyses were used to narrow down prognostic genes and develop an HCC risk model. Using "caret", R separated TCGA-HCC samples into a training risk set and an internal test risk set. As external validation, we used ICGC samples. We employed Kaplan-Meier analysis and receiver operating characteristic (ROC) curve to evaluate the model's clinical efficacy. CIBERSORT and TIMER measured immunocytic infiltration in high- and low-risk populations. Results: TXNRD1 [hazard ratio (HR) =1.477, P<0.001], FTL (HR =1.373, P=0.001), GPX4 (HR =1.650, P=0.004), PRDX1 (HR =1.576, P=0.002), VDAC2 (HR =1.728, P=0.008), OTUB1 (HR =1.826, P=0.002), NRAS (HR =1.596, P=0.005), SLC38A1 (HR =1.290, P=0.002), and SLC1A5 (HR =1.306, P<0.001) were distinguished to build predictive model. In both the model cohort (P<0.001) and the validation cohort (P<0.05), low-risk patients had superior overall survival (OS). The areas under the curve (AUCs) of the ROC curves in the training cohort (1-, 3-, and 5-year AUCs: 0.751, 0.727, and 0.743), internal validation cohort (1-, 3-, and 5-year AUCs: 0.826, 0.624, and 0.589), and ICGC cohort (1-, 3-, and 5-year AUCs: 0.699, 0.702, and 0.568) were calculated. Infiltration of immune cells and immunological checkpoints were also connected with our signature. Treatments with BI.2536, Epothilone.B, Gemcitabine, Mitomycin.C, Obatoclax. Mesylate, and Sunitinib may profit high-risk patients. Conclusions: We analyzed FRGs and CRGs profiles in HCC and established a unique risk model for treatment and prognosis. Our data highlight FRGs and CRGs in clinical practice and suggest ferroptosis and cuproptosis may be therapeutic targets for HCC patients. To validate the model's clinical efficacy, more HCC cases and prospective clinical assessments are needed.

Identification of cuproptosis and ferroptosis-related subgroups and development of a signature for predicting prognosis and tumor microenvironment landscape in hepatocellular carcinoma.

Background: Ferroptosis and cuproptosis play a crucial role in the progression and dissemination of hepatocellular carcinoma (HCC). The primary objective of this study was to develop a unique scoring system for predicting the prognosis and immunological landscape of HCC based on ferroptosis-related genes (FRGs) and cuproptosis-related genes (CRGs). Methods: As the training cohort, we assembled a novel HCC cohort by merging gene expression data and clinical data from The Cancer Genome Atlas (TCGA) database, and Gene Expression Omnibus (GEO) database. The validation cohort consisted of 230 HCC cases taken from the International Cancer Genome Consortium (ICGC) database. Multiple genomic characteristics, such as tumor mutation burden (TMB), and copy number variations were analyzed concurrently. On the basis of the expression of CRGs and FRGs, patients were classified into cuproptosis and ferroptosis subtypes. Then, we constructed a risk model using least absolute shrinkage and selection operator (LASSO) analysis and Cox regression analysis based on ferroptosis and cuproptosis-related differentially expressed genes (DEGs). Patients were separated into two groups according to median risk score. We compared the immunophenotype, tumor microenvironment (TME), cancer stem cell index, and treatment sensitivity of two groups. Results: Three subtypes of ferroptosis and two subtypes of cuproptosis were identified among the patients. A greater likelihood of survival (P<0.05) was expected for patients in FRGcluster B and CRGcluster B. After that, a confirmed risk signature for ferroptosis and cuproptosis was developed and tested. Patients in the low-risk group had significantly higher survival rates than those in the high-risk group, according to our study (P<0.001). There was also a strong correlation between the signature and other variables including immunophenoscore, TMB, cancer stem cell index, immunological checkpoint genes, and sensitivity to chemotherapeutics. Conclusions: Through this comprehensive research, we identified a unique risk signature associated with HCC patients' treatment status and prognosis. Our findings highlight FRGs' and CRGs' significance in clinical practice and imply ferroptosis and cuproptosis may be therapeutic targets for HCC patients.

Soluble expression of recomb inant cMyc, Klf4, Oct4, and Sox2 proteins in bacteria and transduction into living cells.

AIM: To develop a new method to produce recombinant reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, in soluble format with low cost for the generation of induced pluripotent stem cells (iPSCs). METHODS: A short polypeptide sequence derived from the HIV trans-activator of transcription protein (TAT) and the nucleus localization signal (NLS) polypeptide were fused to the N terminus of the reprogramming proteins and they were constructed into pCold-SUMO vector which can extremely improve the solubility of recombinant proteins. Then these vector plasmids were transformed into E. coli BL21 (DE3) Chaperone competent cells for amplification. The solubility of these recombinant proteins was determined by SDS-PAGE and Coomassie brilliant blue staining. The recombinant proteins were purified by Ni-NTA resin and identified by Western blot. The transduction of these proteins into HEK 293T cells were evaluated by immunofluorescence staining. RESULTS: These four reprogramming proteins could be produced in soluble format in pCold-SUMO expression vector system with the assistance of chaperone proteins in bacteria. The proteins were purified successfully with a purity of over 70% with a relative high transduction rate into 293 cells. CONCLUSION: The results in the present study indicate the four important reprogramming proteins, cMyc, Klf4, Oct4, and Sox2, can be produced in soluble format in bacteria with low cost. Our new method thus might be expected to greatly contribute to the future study of iPSCs.

Effects of oleic acid on the corneal permeability of compounds and evaluation of its ocular irritation of rabbit eyes.

OBJECTIVE: To explore the use of oleic acid (OA) in ocular drug delivery. METHODS: Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. The effect of OA on the corneal permeability of drugs was evaluated in vitro, using isolated rabbit corneas by a Franz diffusion cell. The safety of OA was assessed on the basis of corneal hydration level. The ocular irritation of OA was also tested in rabbits in vivo using the Draize eye test. RESULTS: In the presence of OA, at a concentration of 0.02-0.1%, the maximum increase in the apparent permeability coefficient (Papp) was 3.21-, 1.76- and 1.57-fold for rhodamine B, sodium-fluorescein and FITC-dextran of 4 kDa, respectively. However, no significant permeability enhancement of FITC-dextrans of 4, 10, 20 and 40 kDa was found in the presence of OA. It enhanced the corneal penetration of model compounds in a concentration-dependent manner. The Papp values of rhodamine B decreased with increasing concentration of OA, while the Papp values of sodium-fluorescein and FITC-dextrans of 4 kDa increased. The Papp enhanced by 0.1% OA was logarithmically correlated to the molecular weight of model drugs (R(2) = 0.9991). With the 0.02%, 0.05% and 0.1% oleic application, the corneal hydration values were <83%, and Draize scores were <4. CONCLUSION: OA may have potential clinical benefits in improving the ocular drug delivery of both hydrophilic and lipophilic compounds.

The protective effect of vanadium against diabetic cataracts in diabetic rat model.

The present study was designed to investigate the effect of vanadium in alloxan-induced diabetes and cataract in rats. Different doses of vanadium was administered once daily for 8 weeks to alloxan-induced diabetic rats. To know the mechanism of action of vanadium, lens malondialdehyde (MDA), protein carbonyl content, activity of superoxide dismutase (SOD), activities of aldose reductase (AR), and sorbitol levels were assayed, respectively. Supplementation of vanadium to alloxan-induced diabetic rats decreased the blood glucose levels due to hyperglycemia, inhibited the AR activity, and delayed cataract progression in a dose-dependent manner. The observed beneficial effects may be attributed to polyol pathway activation but not decreased oxidative stress. Overall, the results of this study demonstrate that vanadium could effectively reduce the alloxan-induced hyperglycemia and diabetic cataracts in rats.

In vitro evaluation of enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes.

To investigate the enhancing effect of borneol on transcorneal permeation of compounds with different hydrophilicities and molecular sizes. Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. Permeation studies were performed using excised cornea of rabbits by a Franz-type diffusion apparatus. The safety of borneol was assessed on the basis of corneal hydration level and Draize eye test. The application of 0.2% borneol to the cornea increased the apparent permeability coefficient by 1.82-(P<0.05), 2.49-(P<0.05), 4.18-(P<0.05) and 1.11-fold (not significant) for rhodamine B, sodium-fluorescein, FITC-dextrans of 4 and 10 kDa, respectively. No significant permeability enhancement of FITC dextrans of 10, 20 and 40 kDa with borneol was found compared to control. The permeability coefficient enhanced by 0.2% borneol was linear correlated to the molecular weight of model drugs (R(2)=0.9976). With the 0.05%, 0.1% and 0.2% borneol application, the corneal hydration values were <83% and Draize scores were <4. Borneol may improve the transcorneal penetration of both hydrophilic and lipophilic compounds without causing toxic reactions, especially hydrophilic ones. Furthermore, 0.2% borneol can enhance the permeation of hydrophilic compounds with molecular weight ≤4 kDa. Hence, borneol can be considered as a safe and effective penetration enhancer for ocular drug administration.

Preventive effect of danshensu on selenite-induced cataractogenesis in cultured rat lens.

BACKGROUND: To investigate the preventive effect of danshensu on the selenite-induced opacification of cultured rat lenses. METHODS: Isolated lens were divided into three groups with eight lenses in each group. Group I: lenses were incubated with M199 medium alone; Group II: incubated in M199 containing 200 µmol/L sodium selenite; Group III: incubated in M199 containing 200 µmol/L sodium selenite and 500 µmol/L danshensu. Selenite was administered on the third day, and danshensu treatment was from the second to the fifth day. Cataracts development was observed using an inverted microscope, and the lenses were analysed for total anti-oxidative capabilities, mean activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase; levels of reduced glutathione; malondialdehyde; and total sulfhydryl content. RESULTS: All lenses in Group I were clear, whereas all lenses in Group II developed dense vacuolization and opacification. In Group III, 25% lenses revealed minimal vacuolization, and 75% showed no opacification or vacuolization. Total anti-oxidative capabilities and the mean activities of anti-oxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase; levels of glutathione; and total sulfhydryl content were elevated, and the level of malondialdehyde was decreased following treatment with danshensu compared with Group II. CONCLUSION: The anti-oxidative properties of danshensu may play a major role in its contribution to the anticataract effect.

[Relationship between pesticide exposure and adverse pregnancy outcomes among famers: a meta-analysis].

OBJECTIVE: To analyze the relationship between pesticide exposure and adverse pregnancy outcomes in famers. METHODS: A search was conducted to collect the articles about the relationship between pesticide exposure and adverse pregnancy outcomes published worldwide from 1990 to February 2012. Meta-analysis was performed on the collected articles using RevMan 4.2 software. RESULTS: Twelve articles were collected. Compared with the controls, the pesticide-exposed famers showed a combined odds ratio (OR) for spontaneous abortion of 1.52 (95%CI: 1.04 ∼ 2.21; P = 0.03), a combined OR for premature birth of 1.33 (95%CI: 1.09 ∼ 1.61; P = 0.005), a combined OR for dead fetus of 1.22 (95%CI: 1.16 ∼ 1.29; P < 0.01), a combined OR for stillbirth of 1.90 (95%CI: 0.58 ∼ 6.28; P = 0.29), a combined OR for birth defect of 2.02 (95%CI: 0.84 - 4.69; P = 0.12), a combined OR for low birth weight of 1.62 (95%CI: 0.60 ∼ 4.39; P = 0.34), a combined OR for neonatal death of 2.18 (95%CI: 0.54 ∼ 8.88; P = 0.28), and a combined OR for delayed conception of 1.43 (95%CI: 0.93 ∼ 2.18; P = 0.1). Pesticide exposure increased the risks for spontaneous abortion, premature birth, and dead fetus, but was not significantly associated with stillbirth, birth defect, low birth weight, neonatal death, and delayed conception. CONCLUSION: Pesticide exposure can cause adverse pregnancy outcomes in farmers, increasing the risks of spontaneous abortion, premature birth, and dead fetus.

Ursodeoxycholic acid prevents selenite-induced oxidative stress and alleviates cataract formation: In vitro and in vivo studies.

OBJECTIVE: To evaluate the antioxidative and anticataractogenic potential effect of ursodeoxycholic acid (UDCA) on selenite-induced cataract in vitro and in vivo. METHODS: Enucleated rat lenses were incubated in M199 medium alone (Group I), with 200 µM selenite (Group II), or with 200 µM selenite and 500 µM UDCA (Group III). Selenite was administered on the third day and UDCA treatment was from the second to the fifth day. The development of cataracts was observed under an inverted microscope. Total antioxidative capabilities (T-AOC), mean activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx), glutathione reductase (GR) and glutathione S-transferase (GST), levels of reduced glutathione (GSH), malondialdehyde (MDA), and total sulfhydryl content were analyzed in lenticular samples. In vivo, cataracts were induced in 12-day-old pups by single subcutaneous injections of sodium selenite. The test groups received 180 mg/kg bodyweight/day of UDCA intraperitoneally on postpartum days 11-16 or 0.5% UDCA drops four times daily on postpartum days 11-25. RESULTS: In vitro, morphological examination of the lenses revealed dense vacuolization and opacification in Group II, minimal vacuolization in 12.5% of Group III, and no opacification in 87.5% of Group III. In Group I, all lenses were clear. UDCA significantly (p<0.05) restored GSH and total sulfhydryl, and decreased MDA levels. T-AOC and the mean activities of the antioxidant enzymes were elevated following treatment with UDCA. In vivo, 0.5% UDCA drops resulted in only 20% nuclear cataract development and 180 mg/kg of UDCA intraperitoneally led to 50% development, compared to 100% in the control group (p<0.05). CONCLUSIONS: UDCA prevents selenite toxicity and cataractogenesis by maintaining antioxidant status and GSH, protecting the sulfhydryl group, and inhibiting lipid peroxidation in lenses.

Association of glutathione S-transferases polymorphisms (GSTM1 and GSTT1) with senile cataract: a meta-analysis.

PURPOSE: Glutathione S-transferase (GST) polymorphisms have been considered risk factors for the development of senile cataract. However, the results are not consistent. In this study, the authors conducted a meta-analysis to assess the association between GSTM1 and GSTT1 null genotypes and the risk for senile cataract. METHODS: Published literature from PubMed, EMBASE, and other databases were retrieved. All studies evaluating the association between GSTM1/GSTT1 polymorphisms and senile cataract were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using fixed- or random-effects model. RESULTS: Eleven studies on GSTM1 (1871 cases and 1267 controls) and five studies on GSTT1 (1180 cases, 706 controls) were included. Overall analysis showed that the association between GSTM1 null genotype and risk for senile cataract is not statistically significant (OR, 1.39; 95% CI, 0.99-1.94; P = 0.054) and that the association between GSTT1 null genotype and risk for senile cataract is not significant (OR, 1.09; 95% CI, 0.87-1.36; P = 0.454). Subgroup analysis showed that the association between GSTM1 null genotype and risk for senile cataract is statistically significant in Asians (OR, 1.66; 95% CI, 1.03-2.67; P = 0.039) but not in Caucasians (OR, 1.21; 95% CI, 0.74-1.96; P = 0.443). Similar results were observed for the association between GSTT1 null genotype and risk for senile cataract. CONCLUSIONS: The present meta-analysis suggested that GSTM1 and GSTT1 null genotypes are associated with increased risk for senile cataract in Asian populations but not in Caucasian populations. Given the limited sample size, the finding on GST polymorphisms merits further investigation.