Investigation of the assembly of chylomicrons in hamster enterocytes using pluronic-L81 acid as a probe

A major function of the enterocytes is absorption, processing, and export of dietary lipids into the lymphatic system. Pluronic L-81 is a non-ionic hydrophobic surfactant, which specifically inhibits lipid absorption in the intestine when administered in vivo. This compound is therefore an attractive probe to perturb and investigate the molecular and intracellular events in chylomicron assembly in the enterocytes. In the present study pluronic acid was administered to hamsters followed by isolation of the enterocytes and cell fractionation to investigate the effect of pluronic acid on intracellular events in lipid absorption. Four types of diet were administered to hamsters for three weeks; low-fat chow, high-fat chow and each diet with or without added pluronic acid. Sub-cellular fractions of freshly isolated enterocytes were prepared. Consistent with morphological observation, the high fat diet resulted in a three-fold increase in the triacylglycerol (TAG) content of the enterocytes and addition of pluronic acid to either the low fat or the high fat diets resulted in a ten-fold increase in cellular TAG levels. Determination of the mass of TAG and the time course of incorporation of 3H-triolein, administered by gavage, showed that the increased TAG was recovered in the microsomal (endoplasmic reticulum) fraction and the cytosol. In microsomes, increased TAG was recovered mainly in the membrane although there was a significant amount in the microsomal luminal contents. Pluronic acid therefore acts at the level of the endoplasmic reticulum and inhibits the assembly of apo-B48 with chylomicron TAG. The excess TAG is transferred to cytosolic stores.     

MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells

Background

Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis.

Methods

Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays.

Results

We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro.

Conclusions

Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.

     

Validation and comparison of two quantitative real-time PCR assays for direct detection of DMD/BMD carriers

Objectives

To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene.

Design and methods

We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA.

Results

The results showed the gene dosage ratio of 0.99 ± 0.14 and 1.09 ± 0.19 for normal individuals and 0.48 ± 0.06 and 0.50 ± 0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA.

Conclusion

Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.     

Three Markers in Cancerous and Healthy Cells of Patients with Non-Small-Cell Lung Carcinoma (NSCLC)

Introduction: Lung cancer is the most common cause of cancer-related death among males and females. Thediagnosis of lung cancer is of great importance for clinical considerations and follow-up treatment. This study aimedto examine the expression of CEA, LUNX, and CK19 biomarkers in the cancerous and healthy tissues of patientssuffering from NSCLC. Methods: In this study, 30 patients with NSCLCs referring to Masih Daneshvari Hospitalin Tehran were voluntarily selected prior to taking any treatment. A tissue sample from the center and a sample ofhealthy tissues close to the cancerous masses were prepared by a specialist in the bronchoscopy sector and tested usingreal-time RT-PCR. Results: Positive CEA mRNA was observed in cancerous tissues in the center of tumors of 25 outof 30 cases. In the healthy tissue group, the same was found in 10 out of 30 cases (P<0.001). The markers CK19 andLUNX mRNAs showed to be positive in cancerous samples in the center of tumors of 15 and 22 out of 30 cases, and inthe healthy tissue group, the expression was observed in 5 and 4 out of 30 cases, respectively(P<0.001). Conclusion:This study confirms that the aformentioed markers are the ones with a relatively appropriate sensitivity and specificityfor the diagnosis of lung cancer.     

A modified vertical muscle transposition for the treatment of large-angle esotropia due to sixth nerve palsy

Introduction: Multiple different procedures have been proposed to address complete sixth nerve palsy with severe abduction limitation. In this study, we report a modification of the Hummelsheim’s procedure. It is in fact muscle pulley transposition that obviates the need for tenotomy or muscle splitting. For the first time, Muraki and Nishida proposed this technique.

Materials and Methods: Patients with large angle esotropia and abduction limitation of minus four or greater were enrolled. The surgery involved insertion of a polyester monofilament fiber suture through the temporal muscular margin of each vertical rectus muscle at approximately one-third of the width from the edge at 10 mm behind the muscle insertion. We tried to insert sutures away from the vessels of vertical muscles. Then, the vertical muscles were transposed without any tenotomy or splitting and the sutures were secured to the sclera 16 mm from the limbus in supratemporal and infratemporal quadrants. In all of the patients, this transposition was combined with medial rectus recession.

Results: A total of 10 patients were included; all of them had an esotropia with profound abduction deficit (?4 or more). The mean age of patients was 44.2 ± 9.2 years (mean ± standard deviation) (range: 28–57). The mean preoperative deviation was 49.5 ± 9 PD prism diopters (PD) (range: 40–65 PD). The mean preoperative abduction limitation was ?4.8 ± 0.8. The patients were followed for at least 6 months. Postoperative deviation ranged from orthotropia to 12 PD of esotropia and all the patients obtained abduction at least beyond the midline. No vertical ductional disturbances or deviations were developed. The adduction was not compromised in any patient. Anterior segment ischemia did not occur in any patients.

Conclusion: This procedure is comparable to traditional procedures with the advantages of no need to tenotomy or splitting and can be a good alternative to conventional Hummelsheim’s procedure.     

Histopathology study on pulp response to glass ionomers in human teeth

OBJECTIVE: Evaluation of the pulpal response to a resin-modified glass ionomer, a conventional glass ionomer and calcium hydroxide. METHODS AND MATERIALS: Fifty-five deep Class V cavities were lined with Vivaglass Liner, Chembond Superior and Dycal. After seven, 30, and 60 days the teeth were extracted and a histological assessment was performed. RESULTS: There was no statistically significant difference in pulpal response among the three groups for the same time interval (P > 0.05). CONCLUSION: Light-cured glass ionomers have similar advantages to conventional glass ionomers.     

Abdominal fat distribution and carotid atherosclerosis in a general population: a semi-automated method using magnetic resonance imaging

Purpose

Available evidence suggests functional differences in visceral and subcutaneous fat. We investigated the association between quantitative measures of central adiposity with indicators of carotid atherosclerosis including intima-media thickness (IMT) and plaque in a general population using a semi-automated method on magnetic resonance imaging (MRI) data.

Methods

In this cross-sectional study 200 subjects (52 % female), aged 50–77 years, were randomly selected from Golestan Cohort Study. Participants underwent ultrasound examination of carotid arteries and abdominal MRI. Segmentation and calculation of visceral (VFA) and subcutaneous fat area (SFA) were performed on three levels using semi-automated software. Various conventional anthropometric indices were also measured.

Results

Among 191 enrolled subjects, 77 (40 %) participants had IMT ≥0.8 mm. Carotid plaques were detected in 86 (44 %) subjects. In separate multivariate analysis models, unlike SFA and other anthropometric indices, the last tertile of VFA values was associated with at least threefold excess risk for IMT ≥0.8 mm (OR 3.8, 95 % CI 1.36–6.94, p = 0.02). There was no significant difference between mean values of categorized obesity indices in subjects with and without plaque, while participants in the highest tertile of VFA values were demonstrated to have higher risk of more than one plaque (OR 4.57, 95 % CI 1.03–20.11, p = 0.034).

Conclusions

A higher amount of visceral fat, measured by a semi-automated technique using MRI, is associated with increased IMT and having more than one carotid plaque in a general population, while subcutaneous fat measures are poor indicators for identifying carotid atherosclerosis.