MUC-1 aptamer targeted superparamagnetic iron oxide nanoparticles for magnetic resonance imaging of pancreatic cancer in vivo and in vitro experiment

This study aims to explore the ability of magnetic resonance imaging (MRI) in mucin 1 (MUC1) modified superparamagnetic iron oxide nanoparticle (SPION) targeting human pancreatic cancer (PC). The MUC1 target-directed probe was prepared through MUC1 conjugated to SPION using the chemical method to assess its physiochemical characteristics, including hydration diameter, surface charge, and magnetic resonance signal. The cytotoxicity of MUC1-USPION was verified by MTS assay. BxPC-3 was cultured with MUC1-USPION and SPION in different concentrations. The combined condition of the targeted probes and cells were observed through Prussian blue staining. The nude mice model of pancreatic cancer was established to investigate the application of the probe. MRI was performed to determine the intensity of the signal of the transplanted tumor, while immunohistochemistry and Western blot analysis were performed to detect the expression of MUC1 after taking the transplanted tumor specimen. The particle size of the prepared molecular probe was 63.5 ± 3.2 nm, and the surface charge was 10.2 mV. Furthermore, the probe solution could significantly reduce the MRI at T₂, and the magnetic resonance transverse relaxation rate (ΔR²) has a linear relationship with the concentration of iron in the solution. The cell viability of MUC1-USPION in different concentrations revealed no statistical difference, according to the MTS assay. In vitro, the MRI demonstrated decreased T2WI signal intensity in both groups, especially the targeting group. In vivo, MUC1 could selectively accumulate in the nude mice model, and significantly reduce the T₂ signal strength. In subsequent experiments, the expression of MUC1 was high in pancreatic cancer tissues, but low in normal pancreatic tissues, as determined by immunohistochemistry and Western blot analysis. The prepared samples can be combined with pancreatic cancer tissue specificity by in vivo imaging, providing reliable early in vivo imaging data for disease diagnosis.     

Antibody raised against extracellular proteinases ofSporothrix schenckii inS. schenkii inoculated hairless mice

Sporothrix schenckii produces two extracellular proteinases, namely proteinase I and II. Proteinase I is a serine proteinase, inhibited by chymostatin, while proteinase II is an aspartic proteinase, inhibited by pepstatin. Studies on substrate specificity and the effect of proteinase inhibitors on cell growth suggest an important role for these proteinases in terms of fungal invasion and growth. There has, however, been no evidence presented demonstrating thatS. schenckii produces 2 extracellular proteinases in vivo. In order to substantiate the in vivo production of proteinases and to attempt a preliminary serodiagnosis of sporotrichosis, serum antibodies against 2 proteinases were assayed usingS. schenckii inoculated hairless mice. Subsequent to an intracutaneous injection ofS. schenckii to the mouse skin, nodules spontaneously formed and disappeared for a period of 4 weeks. Histopathological examination results were in accordance with the microscopic observations. Micro-organisms disappeared during the fourth week. Serum antibody titers against purified proteinases I and II were measured weekly, using enzyme-linked immunosorbent assay (EIA). As a result, the time course of the antibody titers to both proteinases I and II were parallel to that of macroscopic and microscopic observations in an experimental mouse sporotrichosis model. These results suggest thatS. schenckii produces both proteinases I and II in vivo. Moreover, the detection of antibodies against these proteinases can contribute to a serodiagnosis of sporotrichosis.     

TNF-α-induced exosomal miR-146a mediates mesenchymal stem cell-dependent suppression of urethral stricture

The effective treatment of urethral stricture remains a medical problem. The use of proinflammatory cytokines as stimuli to improve the reparative efficacy of mesenchymal stem cells (MSCs) towards damaged tissues represents an evolving field of investigation. However, the therapeutic benefits of this strategy in the treatment of urethral stricture remain unknown. Here, we enriched exosomes derived from human umbilical cord-derived MSCs pretreated with or without tumor necrosis factor alpha (TNF-α) to evaluate their therapeutic effects in an in vivo model of TGFβ1-induced urethral stricture. Male Sprague-Dawley rats received sham (saline) or TGFβ1 injections to urethral tissues followed by incisions in the urethra. Animals in the TGFβ1 injection (urethral fibrosis) cohort were subsequently injected with vehicle control, or with exosomes derived from MSCs cultured with or without TNF-α. After 4 weeks, rats underwent ultrasound evaluation and, following euthanasia, urethral tissues were harvested for histological and molecular analysis. In vitro, the effects of MSC-derived exosomes on fibroblast secretion of collagen and cytokines were studied by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Exosomes derived from MSCs pretreated with TNF-α were more effective in suppressing urethral fibrosis and stricture than exosomes from untreated MSCs. We found that miR-146a, an anti-inflammatory miRNA, was strongly upregulated in TNF-α-stimulated MSCs and was selectively packaged into exosomes. Moreover, miR-146a-containing exosomes were taken up by fibroblasts and inhibited fibroblast activation and associated inflammatory responses, a finding that may underlie the therapeutic mechanism for suppression of urethral stricture. Inhibition of miR-146a in TNF-α-treated MSCs partially reduced antifibrotic effects and increased the release of proinflammatory factors of exosomes derived from these cells. Together these findings demonstrate that exosomes derived from TNF-α-treated MSCs are of therapeutic benefit in urethral fibrosis, suggesting that this strategy may have utility as an adjuvant therapy in the treatment of urethral stricture diseases.     

GABA concentrations in the striatum following repetitive cerebral ischemia

GABAergic neurons in the striatum are very sensitive to the effects of ischemia. The progressive decline in striatal GABA following transient forebrain ischemia in gerbils may be secondary to either a decreased production or an increase in reuptake mechanisms or both. The current experiment was designed to evaluate release of GABA by stimulation with K+ or inhibition of its uptake with nipecotic acid or their combination (K+ nipecotic) after repetitive forebrain ischemia in gerbils by in-vivo microdialysis on Days 1, 3, 5, and 14 following the insult. Infusion of nipecotic acid or potassium chloride, resulted in a significant increase in extracellular GABA. This response was significantly decreased in the post-ischemic animals. The synergistic effect of increased GABA concentrations by the infusion of nipecotic acid+potassium chloride seen in the controls was not evident in the post-ischemic animals. In conclusion, though there is a reduction in the extracellular GABA concentrations in the first week following an ischemic insult, restorative mechanisms are operative in the second week as seen by the increasing GABA concentrations.     

Hepatitis C virus genotypes in patients with chronic hepatitis C infection in southern Iran from 2016 to 2019

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The treatment of HCV infection has become more complicated due to various genotypes and subtypes of HCV. The treatment of HCV has made significant advances with direct-acting antivirals. However, for the choice of medicine or the combination of drugs for hepatitis C, it is imperative to detect and discriminate the crucial HCV genotypes. The main objective of this study was to determine the pattern of circulating HCV genotypes in southern Iran, from 2016 until 2019. The other aim of the study was to determine possible associations of patients’ risk factors with HCV genotypes. A total of 803 serum samples were collected in 4 years (2016–2019) from patients with HCV antibody positive results. A total of 728 serum samples were HCV-RNA positive. The prevalence of HCV genotypes was detected using the genotype-specific RT-PCR test for serum samples obtained from 615 patients. The HCV genotype 1 (G1) was the most prevalent (48.8%) genotype in the area, with G1a, G1b, and mixed G1a/b representing 38.4%, 10.1%, and 0.3%, respectively. Genotype 3a was the next most prevalent (47.2%). Mixed genotypes 1a/3a were detected in 22 (3.6%) and finally G4 was found in 3 (0.5%) patients. The other HCV genotypes were not detected in any patient. Genotype 1 (1a and 1b alone, 1a/1b and 1a/3a coinfections) is the most prevalent HCV genotype in southern Iran. HCV G1 shows a significantly higher rate in people under 40 years old.     

Identification of potentially critical genes in the development of heart failure after ST-segment elevation myocardial infarction (STEMI)

Heart failure (HF) remains a common complication after acute ST-segment elevation myocardial infarction (STEMI). Here, we aim to identify critical genes related to the developed HF in patients with STEMI using bioinformatics analysis. The microarray data of GSE59867, including peripheral blood samples from nine patients with post-infarct HF and eight patients without post-infarct HF, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) between HF and non-HF groups were screened by LIMMA package. Functional enrichment analyses of DEGs were conducted, followed by construction of a protein-protein interaction (PPI) network. The dynamic messenger RNA (mRNA) level of the hub genes during the follow-up was analyzed to further elucidate their role in HF development. A total of 58 upregulated and 75 downregulated DEGs were screen out. They were mainly enriched in biological processes about inflammatory response, extracellular matrix organization, response to cAMP, immune response, and positive regulation of cytosolic calcium ion concentration. Pathway analysis revealed that the DEGs were also involved in hematopoietic cell lineage, pathways in cancer, and extracellular matrix-receptor interaction. In the PPI network consisting of 58 nodes and 72 interactions, CXCL8 (degree = 15), THBS1 (degree = 8), FOS (degree = 7), and ITGA2B (degree = 6) were identified as the hub genes. In the comparison of patients with and without post-infarct HF, the mRNA level of these hub genes were all higher within 30 days but reached similar at 6 months after STEMI. In conclusion, CXCL8, THBS1, FOS, and ITGA2B may play important roles in the development of HF after acute STEMI.     

E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.     

Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF(KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumor growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228.These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.