A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Diterpenoids from Torreya grandis and their cytotoxic activities.

Eight previously undescribed diterpenoids, along with eleven previously reported analogues, were obtained from the supercritical CO2 extracts of Torreya grandis aril. The structures of these compounds were elucidated based on HRESIMS, NMR, ECD, and single-crystal X-ray diffraction data. In the MTT assay, compound 18 exhibited significant inhibitory effects on two human colon cancer cell lines, HT-29 and HCT 116 cells, with IC50 values of 7.37 µM and 6.55 µM, respectively. It was found that compound 18 induced apoptosis and significantly inhibited the migration of HCT 116 colon cancer cells in a concentration-dependent manner.

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Cytokinin and indole-3-acetic acid crosstalk is indispensable for silicon mediated chromium stress tolerance in roots of wheat seedlings.

The rising heavy metal contamination of soils imposes toxic impacts on plants as well as other life forms. One such highly toxic and carcinogenic heavy metal is hexavalent chromium [Cr(VI)] that has been reported to prominently retard the plant growth. The present study investigated the potential of silicon (Si, 10 µM) to alleviate the toxicity of Cr(VI) (25 µM) on roots of wheat (Triticum aestivum L.) seedlings. Application of Si to Cr(VI)-stressed wheat seedlings improved their overall growth parameters. This study also reveals the involvement of two phytohormones, namely auxin and cytokinin and their crosstalk in Si-mediated mitigation of the toxic impacts of Cr(VI) in wheat seedlings. The application of cytokinin alone to wheat seedlings under Cr(VI) stress reduced the intensity of toxic effects of Cr(VI). In combination with Si, cytokinin application to Cr(VI)-stressed wheat seedlings significantly minimized the decrease induced by Cr(VI) in different parameters such as root-shoot length (10.8% and 13%, respectively), root-shoot fresh mass (11.3% and 10.1%, respectively), and total chlorophyll and carotenoids content (13.4% and 6.8%, respectively) with respect to the control. This treatment also maintained the regulation of proline metabolism (proline content, and P5CS and PDH activities), ascorbate-glutathione (AsA-GSH) cycle and nutrient homeostasis. The protective effect of Si and cytokinin against Cr(VI) stress was minimized upon supplementation of an inhibitor of polar auxin transport- 2,3,5-triiodobenzoic acid (TIBA) which suggested a potential involvement of auxin in Si and cytokinin-mediated mitigation of Cr(VI) toxicity. The exogenous addition of a natural auxin - indole-3-acetic acid (IAA) confirmed auxin is an active member of a signaling cascade along with cytokinin that aids in Si-mediated Cr(VI) toxicity alleviation as IAA application reversed the negative impacts of TIBA on wheat roots treated with Cr(VI), cytokinin and Si. The results of this research are also confirmed by the gene expression analysis conducted for nutrient transporters (Lsi1, CCaMK, MHX, SULT1 and ZIP1) and enzymes involved in the AsA-GSH cycle (APX, GR, DHAR and MDHAR). The overall results of this research indicate towards possible induction of a crosstalk between cytokinin and IAA upon Si supplementation which in turn stimulates physiological, biochemical and molecular changes to exhibit protective effects against Cr(VI) stress. Further, the information obtained suggests probable employment of Si, cytokinin and IAA alone or combined in agriculture to maintain plant productivity under Cr(VI) stress and data regarding expression of key genes can be used to develop new crop varieties with enhanced resistance against Cr(VI) stress together with its reduced load in seedlings.

Exploration of antiproliferative potential of modified triazole-benzohydrazone scaffold: Multitarget approach.

A novel series of triazole-benzohydrazone hybrids was efficiently designed and synthesized as antiproliferative agents, targeting different kinases. All compounds were screened via the National Cancer Institute (NCI) against 60 cancer cell lines, where compounds 16, 17, and 18 exhibited growth inhibition percent (GI%) of various leukemia subpanels with values of 70.33%, 64.13%, and 76.03%, respectively. Compound 18 showed broad-spectrum antiproliferative efficacy toward most cancer cells, with outstanding potency regarding melanoma (MALME-3M GI% = 101.82%) and breast cancer cell lines (MCF7 GI% = 85.87%), while proving safe toward the WI-38 normal cell line, compared to doxorubicin. Multikinase investigation including vascular endothelial growth factor receptor 2 (VEGFR-2), mesenchymal epithelial transition factor (c-Met), proto-oncogene B-Raf, mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, and phosphoinositide 3-kinase was accomplished to reveal its plausible mechanism of action, giving the ultimate potency against both VEGFR-2 and c-Met with IC50 values of 0.055 and 0.042 µM, respectively, while displaying moderate to good inhibition concerning the remaining kinases. DNA binding capability was excluded using the methyl green colorimetric assay. Further, it exhibited both early and late apoptotic induction by about 16- and 9.4-fold over the control, respectively, triggering cell cycle arrest in the G2/M phase. Physicochemical properties and bioavailability radar plot inferred drug-likeness characteristics for compound 18. The molecular docking study assessed the binding pattern with the active sites of c-Met and VEGFR-2.

Berberine alleviates contrast-induced nephropathy by activating Akt/Foxo3a/Nrf2 signalling pathway.

Contrast-induced nephropathy (CIN) is a condition that causes kidney damage in patients receiving angiography with iodine-based contrast agents. This study investigated the potential protective effects of berberine (BBR) against CIN and its underlying mechanisms. The researchers conducted both in vivo and in vitro experiments to explore BBR's renal protective effects. In the in vivo experiments, SD rats were used to create a CIN model, and different groups were established. The results showed that CIN model group exhibited impaired renal function, severe damage to renal tubular cells and increased apoptosis and ferroptosis. However, BBR treatment group demonstrated improved renal function, decreased apoptosis and ferroptosis. Similar results were observed in the in vitro experiments using HK-2 cells. BBR reduced ioversol-induced apoptosis and ferroptosis, and exerted its protective effects through Akt/Foxo3a/Nrf2 signalling pathway. BBR administration increased the expression of Foxo3a and Nrf2 while decreasing the levels of p-Akt and p-Foxo3a. In conclusion, this study revealed that BBR effectively inhibited ioversol-induced apoptosis and ferroptosis in vivo and in vitro. The protective effects of BBR were mediated through the modulation of Akt/Foxo3a/Nrf2 signalling pathway, leading to the alleviation of CIN. These findings suggest that BBR may have therapeutic potential for protecting against CIN in patients undergoing angiography with iodine-based contrast agents.

Preoperative Contrast Examinations Help Determine the Appropriate Cervical Approach for Congenital Gross Type C Esophageal Atresia: A Report of Two Cases.

BACKGROUND Kluth demonstrated that esophageal atresia/tracheoesophageal fistula (EA/TEF) has several anatomical variations and thus requires a preoperative imaging study to determine the surgical strategy. We routinely perform a contrast examination with iodixanol to assess the location of the TEF and the upper end of the esophageal pouch to determine the most appropriate approach. We herein present two cases of type C EA/TEF who successfully underwent radical surgery by a cervical approach based on the information from the contrast examination. CASE REPORT Case 1 was a Japanese boy suspected of type C EA/TEF after birth. A contrast examination with iodixanol showed that a TEF was at the second thoracic vertebra (Th2), as was the upper end of the esophageal pouch. Thus, the patient underwent esophago-esophageal anastomosis and TEF ligation using a cervical approach; the postoperative course was uneventful. Case 2 was also a Japanese boy suspected of type C EA/TEF. A contrast examination showed that the TEF was at Th1-2, as was the upper end of the esophageal pouch. Thus, the patient underwent esophago-esophageal anastomosis and TEF ligation using a cervical approach. The patient suffered from congenital tracheal stenosis and required tracheoplasty. However, there were no apparent complications after the surgery. CONCLUSIONS Here, we used the imaging information to adopt the cervical approach in type C EA/TEF cases and concluded that routine preoperative contrast examinations helped assess the TEF location and upper end of the esophageal pouch without significant complications.

Ioxaglate Versus IoDixanol for the Prevention of Contrast-Induced Nephropathy: The IDPC Trial.

BACKGROUND: Despite the potential benefits of percutaneous procedures for the assessment and treatment of coronary artery disease, these interventions require the use of iodine contrast, which might lead to contrast-induced nephropathy (CIN) and increased risk of dialysis and major adverse cardiac events (MACE). AIMS: We sought to compare two different iodine contrasts (low vs. iso-osmolar) for the prevention of CIN among high-risk patients. METHODS: This is a single-center, randomized (1:1) trial comparing consecutive patients at high risk for CIN referred to percutaneous coronary diagnostic and/or therapeutic procedures with low (ioxaglate) vs. iso-osmolarity (iodixanol) iodine contrast. High risk was defined by the presence of at least one of the following conditions: age >70 years, diabetes mellitus, non-dialytic chronic kidney disease, chronic heart failure, cardiogenic shock, and acute coronary syndrome (ACS). The primary endpoint was the occurrence of CIN, defined as a >25% relative increase and/or >0.5 mg/dL absolute increase in creatinine (Cr) levels compared with baseline between the 2nd and 5th day after contrast media administration. RESULTS: A total of 2,268 patients were enrolled. Mean age was 67 years. Diabetes mellitus (53%), non-dialytic chronic kidney disease (31%), and ACS (39%) were highly prevalent. The mean volume of contrast media was 89 ml ± 48.6. CIN occurred in 15% of all patients, with no significant difference regarding the type of contrast used (iso = 15.2% vs. low = 15.1%, P>.99). Differences were not observed in specific subgroups such as diabetics, elderly, and ACS patients. At 30-day follow-up, 13 patients in the iso-osmolarity group and 11 in low-osmolarity group required dialysis (P =.8). There were 37 (3.3%) deaths in the iso-osmolarity cohort vs. 29 (2.6%) in the low-osmolarity group (P =.4). CONCLUSION: Among patients at high risk for CIN, the incidence of this complication was 15%, and independent of the use of low- or iso-osmolar contrast.

Natural Compounds with BMI1 Promoter Inhibitory Activity from Mammea siamensis and Andrographis paniculata.

A new coumarin derivative (1) and 30 known compounds were isolated from Mammea siamensis and Andrographis paniculata, guided by B cell-specific Moloney murine leukemia virus insertion region 1 (BMI1) promoter inhibitory activity. Among the isolated compounds, 15 compounds showed BMI1 promoter inhibitory activity, and five compounds were found to be cytotoxic. 14-Deoxy-11,12-dehydroandrographolide (18) was highly cytotoxic to DU145 cells with an IC50 value of 25.4 µM. Western blotting analysis of compound 18 in DU145 cells suggested that compound 18 suppresses BMI1 expression.

Lu3+-based nanoprobe for virtual non-contrast CT imaging of hepatocellular carcinoma.

Transcatheter arterial chemoembolization (TACE), the mainstream treatment for hepatocellular carcinoma (HCC), is a method of blocking tumor blood vessels with a mixture of lipiodol and chemotherapeutics. And the contrast-enhanced computed tomography (CT) is the commonly used way for follow-up of HCC after TACE. However, it is noteworthy that when lipiodol deposition plays an embolic effect, it also produces high-density artifacts in CT images. These artifacts usually conceal the enhancement effect of iodine contrast agents. As a result, the residual region is difficult to be visualized. To overcome this obstacle, we developed one kind of Lu3+/Gd3+ doped fluoride nanoprobe modified with Dp-PEG2000 to realize CT/MRI dual-modality imaging of HCC. Compared with lipiodol or ioversol, the obtained PEGylated product LG-PEG demonstrated a greater density value in high keV CT images. In vitro experiments showed the lipiodol artifacts can be removed in virtual non-contrast (VNC) imaging, but the density of ioversol was also removed at the same time. However, the LG-PEG synthesized in this work can still maintain a high density in VNC imaging, which indicates that LG-PEG can exploit its advantages to the full in VNC imaging. Furthermore, LG-PEG successfully exerted tumor enhancement effects in the in vivo VNC images of HCC with lipiodol deposition. In addition, LG-PEG exhibited a strong T2 enhancement effect with low biological toxicity and less side-effect on the main organ and blood. Thus, the LG-PEG reported in this research can serve as an effective and safe VNC contrast agent for HCC imaging after TACE.

Iopromide and Iodixanol in the Development of Postoperative Contrast Nephropathy in Patients with Renal Insufficiency: A Meta-Analysis.

In order to compare the effects of iopromide and isoxazole on postoperative contrast-induced nephropathy in patients with renal insufficiency, the paper searches for randomized controlled trials and retrospective cohort studies comparing the effects of iopromide and iodixanol on renal function in patients with renal insufficiency after surgery. The data are extracted from eligible studies. We tried to assess the incidence of contrast-agent nephropathy, preoperative and postoperative serum creatinine indicators, and mortality. This paper includes 8 studies with a total of 1243 patients. The incidence of contrast-induced nephropathy in the iopromide group is higher than that in the iodixanol group, and there is no significant difference between the two groups in postoperative mortality and preoperative serum creatinine expression. Sensitivity analysis and funnel chart show that our research is robust and has low publication bias. Our research shows that in patients with renal insufficiency, the incidence of contrast-medium nephropathy in the iopromide group is higher than that in the iodixanol group. Iodixanol is safer and has less effect on patients' serum creatinine levels.

Reassessment of the Proteomic Composition and Function of Extracellular Vesicles in the Seminal Plasma.

Seminal plasma contains a high concentration of extracellular vesicles (EVs). The heterogeneity of small EVs or the presence of nonvesicular extracellular matter (NV) pose major obstacles in understanding the composition and function of seminal EVs. In this study, we employed high-resolution density gradient fractionation to accurately characterize the composition and function of seminal EVs and NV. We found that the seminal EVs could be divided into 3 different subtypes-namely, high-density EV (EV-H), medium-density EV (EV-M), and low-density EV (EV-L)-after purification using iodixanol, while NV was successfully isolated. EVs and NV display different features in size, shape, and expression of some classic exosome markers. Both EV-H and NV could markedly promote sperm motility and capacitation compared with EV-M and EV-L, whereas only the NV fraction induced sperm acrosome reaction. Proteomic analysis results showed that EV-H, EV-M, EV-L, and NV had different protein components and were involved in different physiological functions. Further study showed that EV-M might reduce the production of sperm intrinsic reactive oxygen species through glutathione S-transferase mu 2. This study provides novel insights into important aspects of seminal EVs constituents and sounder footing to explore their functional properties in male fertility.

Development and Application of a Liquid Chromatography-Mass Spectrometry Method for Residual Iodixanol Quantification in AAV-Based Gene Therapy Product Development.

Adeno-associated viruses (AAVs) are nonenveloped viruses that have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanol-based density gradient is one of the widely used purification methods for serotype-independent AAVs. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed. An analytical assay with high sensitivity is essential for the detection of residual iodixanol to ensure the safety of AAV products. We developed a liquid chromatography-mass spectrometry method with the limit of quantification of 0.01 µg/mL for residual iodixanol measurement in AAVs. The method also demonstrated linearity over four orders of magnitude that allows quantifying a high iodixanol concentration in in-process samples with excellent recovery and accuracy. In addition, we further explored a highly efficient purification method for removal of the residual iodixanol, to minimize the safety concern from iodixanol as a process impurity.

Streak artefacts in Computed Tomography Urography: Comparative evaluation between two different iodinated contrast media.

INTRODUCTION: The objective of this study was to compare the frequency and entity, in computed tomography (CT) urography, of streak artefacts on the urinary tract generated by two contrast agents with a different iodine concentration and osmolarity. METHODS: Computed tomography scans including an excretory renal phase, performed on adult subjects in the period May-July 2020, were retrospectively evaluated in consensus by three expert radiologists, to detect any streak artefacts located in the urinary tract. Patients were administered either 1.6 mL/kg of Iodixanol 320 mgI/mL or 1.3 mL/kg of Iomeprol 400 mgI/mL. RESULTS: In total, 144 CT scans were analysed, subdivided into two groups administered either Iodixanol (71/144 (49.3%) patients) or Iomeprol (73/144 (50.7%) patients). In 41% cases, no beam hardening artefacts were found; among these, 12/59 (20.3%) patients had received Iodixanol and 47/59 (79.7%) Iomeprol. In the Iodixanol group, the mean contrast density on the renal pelvis was 2565.6 HU and streak artefacts occurred in 59/71 cases (83.1%); in 33/59 (55.9%) cases, the artefacts were marked, and in 26/59 (44.1%) minimal. In the Iomeprol group, the mean contrast density on the renal pelvis was 1666 HU and streak artefacts occurred in 26/73 cases (35.6%); in 7/26 (27%) cases, the artefacts were marked and in 19/26 (73%) minimal. CONCLUSION: The study data demonstrate a significant difference in the attenuation values of iodine urine in the excretory system between the Iodixanol and Iomeprol group. Iodixanol induced a higher frequency and burden of artefacts, compared to Iomeprol.

Pulmonary lesions: correlative study of dynamic triple-phase enhanced CT perfusion imaging with tumor angiogenesis and vascular endothelial growth factor expression.

BACKGROUND: To investigate value of the quantitative perfusion parameters of dynamic triple-phase enhanced CT in differential diagnosis of pulmonary lesions, and explore the correlation between perfusion parameters of lung cancer with microvessel density (MVD) and vascular endothelial growth factor (VEGF). METHODS: 73 consecutive patients with lung lesions who successfully underwent pre-operative CT perfusion examination with dynamic triple-phase enhanced CT and received a final diagnosis by postoperative pathology or a clinical follow-up. The cases were divided into malignant and benign groups according to the pathological results. CT perfusion parameters, such as Median, Mean, Standard deviation (Std), Q10, Q25, Q50, Q75, Q90 of pulmonary artery perfusion (PAP), bronchial artery perfusion (BAP), perfusion index (PI) and arterial enhancement fraction (AEF) were obtained by performing computed tomography perfusion imaging (CTPI). Computed tomography perfusion (CTP) parameters were compared between malignant and benign lesions. The receiver operating characteristic (ROC) curve was used to assess the diagnostic efficiency of CTP parameters in diagnosing malignant lesions. The correlations between CTP parameters with MVD and VEGF were analysed in 36 lung cancer patients who had extra sections be used for immunohistochemistry staining of CD34 and VEGF. RESULTS: BAP (Mean, Std, Q90) and PI Std of benign lesions were higher than malignant lesions (p < 0.05), and PAP (Q10, Q25), PI (Median, Mean, Q10, Q25, Q50) of malignant lesions were higher than the benign (p < 0.05). The area under the ROC curve of PI Mean, PI Q10 and PI Std was 0.722 (95% CI = [0.595-0.845]), 0.728 (95% CI = [0.612-0.844]) and 0.717 (95% CI = [0.598-0.835]) respectively. Partial perfusion parameters of BAP and AEF Q10 were positively correlated with MVD (p value range is < 0.001-0.037, ρ value range is 0.483-0.683), and partial perfusion parameters of PI were negatively correlated with MVD (p value range is 0.001-0.041,ρvalue range is - 0.523-- 0.343). Partial perfusion parameters of BAP and AEF Q10 were positively correlated with VEGF (p value range is 0.001-0.016, ρvalue range is 0.398-0.570), meanwhile some perfusion parameters of PAP and PI were negatively correlated with VEGF (p value range is 0.001-0.040, ρ value range is - 0.657-0.343). CONCLUSIONS: Quantitative parameters of dynamic triple-phase enhanced CT can provide diagnostic basis for the differentiation of lung lesions, and there were connection with tumor angiogenesis and vascular endothelial growth factor expression.

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation.

Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

The contrast agent 2,3,5-triiodobenzoic acid (TIBA) induces cell death in tumor cells through the generation of reactive oxygen species.

The 2,3,5-triiodobenzoic acid (TIBA) is an iodine contrast agent used for visualization of tissue in X-ray techniques. However, TIBA induces physiological complications like increase in oxygen reactive species (ROS), and consequently, contrast-induced nephropathies. TIBA's antitumor activity was demonstrated in lung cancer, but the subcellular mechanisms involving its activity in tumor cells are still unknown. Thus, the objective of this work was evaluate whether the anti-tumor activity of TIBA involves ROS increase, in tumor lines of non-small cell lung cancer (H460), chronic myeloid leukemia (K562), and its cytotoxicity in normal renal epithelial (VERO). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay was used for evaluation of cell viability, the H2DCFDA (cell-permeant 2',7'-dichlorodihydrofluorescein diacetate) fluorescent probe to evaluate ROS induction, cell cycle analysis was performed using flow cytometry to measure cell death, and immunofluorescence with annexin/7-AAD (7-amino-actinomycin D), to assess the association of cell death with the ROS generation. TIBA decreases cell viability in a dose-dependent manner for the H460 and K562. However, VERO cells showed less response to the drug, with 70% viable cells after 72 h of treatment in the highest concentration of the drug. While the tumor cells with only 20% viable cells. Besides, tumor cells exhibited higher DNA fragmentation, compared to the renal line (VERO with 5% of fragmented DNA, H460 with 26%, and 56% in K562). Finally, TIBA-induced ROS increase and apoptosis in all lines, which is significantly decreased after treatment with the antioxidant N-acetyl-cysteine (NAC). These data demonstrate the relationship between the increased cellular oxidative stress and the anti-tumor action of the TIBA.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 µl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Circulating Exosomal miR-1-3p from Rats with Myocardial Infarction Plays a Protective Effect on Contrast-Induced Nephropathy via Targeting ATG13 and activating the AKT Signaling Pathway.

Rationale: With the widespread development of the interventional technique for cardiovascular diseases and the widespread use of contrast medium (CM), the incidence of contrast-induced nephropathy (CIN) has been increasing, which is associated with poor prognosis for cardiovascular diseases. This study aims to explore the effect of circulating exosomal microRNA from patients with myocardial infarction (MI) on CIN and related molecular mechanism. Methods: A rat MI model was established by ligating the left anterior descending coronary artery. Circulating exosomes were isolated from control (Exo-NC) and MI rats (Exo-MI) using a commercial kit. The in vivo and in vitro models of CIN were created using iodixanol. Reverse transcription quantitative PCR (RT-qPCR) was utilized to detect the expression of miR-1-3p. Western blot (WB) was used to detect the expression of exosomal surface markers, and apoptosis-related and autophagy-related proteins. The apoptosis rate was examined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry (FC). Transmission electron microscopy (TEM) was utilized to observe the exosomes and autophagosomes. Rat kidney injury was assessed by hematoxylin and eosin (H&E) staining and kidney injury molecule-1 (KIM-1) immunohistochemical staining. Renal function of rats was assessed by detecting the levels of blood urea nitrogen (BUN) and serum creatinine (Cr). The dual luciferase reporter assay was performed to identify the target gene of miR-1-3p. Results: The treatment of CM induced NRK-52E cell damage, which manifested as enhanced cell autophagy and enhanced apoptosis. The Exo-MI treatment significantly inhibited the CM-induced autophagy and apoptosis of NRK-52E cells. Furthermore, the Exo-MI treatment increased the Bcl-2 expression, but decreased the Bax expression and the ratio of LC3II/LC3I. Furthermore, the results of the TUNEL staining and FC showed that Exo-MI can reduce apoptotic rate. Through TEM, it was found that Exo-MI reduced the number of autophagosomes in NRK-52E cells. The rescue experiments revealed that the function of Exo-MI is to inhibit the CM-induced autophagy and apoptosis of NRK-52E cells, which can be inhibited by the miR-1-3p inhibitor. Furthermore, it was found that the overexpression of miR-1-3p can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. Through dual luciferase reporter assay, ATG13 was found to be the target of miR-1-3p. In addition, the overexpression of miR-1-3p significantly reversed the CM-induced decrease in phosphorylation level of AKT. Furthermore, ATG13 silencing can also inhibit the CM-induced autophagy and apoptosis of NRK-52E cells. In vivo, Exo-MI significantly alleviated the renal injury, reduced the renal fibrosis, and improved the renal function of CIN rats. Conclusion: The circulating exosomal miR-1-3p after MI inhibited the CM-induced apoptosis and autophagy of renal tubular epithelial cells, and improved the renal function of rats by targeting ATG13 and activating the AKT signaling pathway.

Isolation and characterization of extracellular vesicle subpopulations from tissues.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.