Gene delivery from supercharged coiled-coil protein and cationic lipid hybrid complex

A lipoproteoplex comprised of an engineered supercharged coiled-coil protein (CSP) bearing multiple arginines and the cationic lipid formulation FuGENE HD (FG) was developed for effective condensation and delivery of nucleic acids. The CSP was able to maintain helical structure and self-assembly properties while exhibiting binding to plasmid DNA. The ternary CSP·DNA(8:1)·FG lipoproteoplex complex demonstrated enhanced transfection of β-galactosidase DNA into MC3T3-E1 mouse preosteoblasts. The lipoproteoplexes showed significant increases in transfection efficiency when compared to conventional FG and an mTat·FG lipopolyplex with a 6- and 2.5-fold increase in transfection, respectively. The CSP·DNA(8:1)·FG lipoproteoplex assembled into spherical particles with a net positive surface charge, enabling efficient gene delivery. These results support the application of lipoproteoplexes with protein engineered CSP for non-viral gene delivery.     

Effect of S-methylisothiourea,an inducible nitric oxide synthase inhibitor,in joint pain and pathology in surgically induced model of osteoarthritis

The aim of the present study was to evaluate in vivo modulatory effect of S-methylisothiourea (SMT), a preferential inhibitor of inducible nitric oxide synthase (iNOS) on pain and pathology in the surgical model of osteoarthritis (OA) in rats. The OA was produced by the anterior cruciate ligament transection (ACLT) and medial meniscectomy (MMx) of right knee. SMT was administered 1 day prior to the production of OA and continued up to day 42 postoperation. Mechanical hyperalgesia, thermal hyperalgesia, tail flick latency after repeated flexion and extension of OA knee and knee diameter of right knee were determined at weekly intervals. Serum levels of IL-1β, TNF-α and nitrite concentration were determined at the end of the experiment. Glycosaminoglycan (GAG) content, collagen content and histopathological evaluation of articular cartilage were also determined at the end of the experiment. SMT reduced mechanical hyperalgesia and the serum levels of IL-1β, TNF-α and nitrite. Further, SMT reduced the loss of GAG from articular cartilage. Microscopically, SMT reduced the severity of the cartilage lesion. The results indicate the effectiveness of SMT in attenuating the pain and pathology of experimental OA phase by reducing the production of nitric oxide and interleukin-1β and tumor necrosis factor-α, which are known to play a major role in the pathophysiology of OA.     

Identification of seven novel germline mutations in the human E-cadherin (CDH1) gene

Hereditary diffuse gastric cancer (HDGC) is a cancer predisposition syndrome caused by germline mutation of the gene encoding the tumour-suppressor E-cadherin (CDH1). We describe the search for CDH1 mutations in 36 new diffuse gastric cancer families. All 16 CDH1 exons, neighbouring intronic sequence and an essential promoter region were screened by DNA sequencing. We detected nine different mutations, seven of which were novel. Of the seven novel mutations, five were identified in families who met the IGCLC clinical criteria for HDGC. Two mutations resulted in a premature stop codon and truncation of the protein. Three mutations affected splice sites; two of the splice-site mutations were shown by RT-PCR to disturb normal CDH1 splicing, while the third splice-site mutation was present in two unrelated HDGC families. The remaining two mutations resulted in amino acid substitutions and impaired the ability of E-cadherin protein to form cellular aggregates and suppress invasion in vitro. Together with the occurrence of extra-gastric tumours such as lobular breast and colorectal cancer, these findings further extend the types of CDH1 mutations and the spectrum of tumours associated with HDGC.     

The hepatic veins: Anatomy and classification on single slice spiral CT in North Indian population

AimsTo find out the normal pattern of hepatic veins in the North Indian population and to categorize them.MethodsThe present study was conducted on 100 patients whose spiral CT abdomen was performed for various medical conditions in the department of radiodiagnosis.ResultsFour categories were recognized. Category-1, when right hepatic vein drains independently into the inferior vena cava whereas middle and left hepatic veins join together to form a common trunk before draining into the inferior vena cava. It was observed in 74% patients. Category-2 was observed in 2% patients, where right & middle hepatic veins join to form a common trunk and left hepatic vein drain independently into the inferior vena cava. Category-3 was observed in 21% patients, where all the three major hepatic veins drain independently into the inferior vena cava. Category-4 was observed in 3% patients, where all the three major hepatic veins join together to form a common trunk before draining into the inferior vena cava.ConclusionsCategory-1 is the most common pattern of major hepatic vein drainage found in the North Indian population. The present study also concluded that single right, middle and left hepatic vein is the most common pattern of hepatic veins present in the North Indian population. Caudate lobe is drained by more than one vein in majority of North Indians. Also superomedial vein, right accessory vein and inferior right hepatic vein are the most common accessory veins present in the North Indian population.     

Nano-TiO2 particles impair adhesion of airway epithelial cells to fibronectin

Titanium dioxide engineered nanoparticles (nano-TiO₂) are widely used in the manufacturing of a number of products. Due to their size (<100 nm), when inhaled they may be deposited in the distal lung regions and damage Clara cells. We investigated the mechanisms by which short-term (1-h) incubation of human airway Clara-like (H441) cells to nano-TiO₂ (6 nm in diameter) alters the ability of H441 cells to adhere to extracellular matrix. Our results show that 1 h post-incubation, there was a 3-fold increase of extracellular H₂O₂, increased intracellular oxidative stress as demonstrated by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) oxidation, and a 5-fold increase of phosphor-ERK1/2 as measured by Western blotting. These changes were accompanied by a 25% decrease of H441 adherence to fibronectin (p < 0.05 compared to vehicle incubated H441 cells). Pretreatment with the ERK1/2 inhibitor U0126 for 3 h, partially prevented this effect. In conclusion, short-term exposure of H441 cells to nano-TiO₂ appears to reduce adherence to fibronectin due to oxidative stress and activation of ERK1/2.     

Serum immunoglobulin G subclass responses in different phases of hepatitis E virus infection

To investigate the specific immunoglobulin (Ig) G subclass responses in patients with hepatitis E virus (HEV) infection, an open reading frame 2 (ORF2) protein based enzyme‐linked immunosorbant assay was used to measure antibody levels in sera obtained at different phases of infection. Sera were collected at 2–31 days and at 6 months after the onset of symptoms corresponding to the acute (n = 48, 100% IgM‐positive) and convalescent (n = 17/48, 53% IgM‐positive) phases of infection, respectively. IgM‐negative sera from 61 individuals infected at least ≥6 months ago (prior exposure) were also tested. IgG1, IgG2, IgG3, and IgG4 antibodies were detected in 100%, 6%, 56%, and 4% of acute phase sera, respectively, and in 100%, 0%, 0%, and 65% of convalescent phase sera, respectively. IgG1 antibody levels were significantly higher than those of the other detectable subclasses of IgG in the acute and convalescent sera (P < 0.05). The IgG3 antibodies in six acute phase patients were replaced by IgG4 antibodies in the convalescent phase of infection. Patients with prior exposure to HEV had low total IgG antibody titers and decreased IgG1 seropositivity compared with those in the acute and convalescent phases. IgG1 was the only major subclass of antibody to be detected in all the three phases of infection. Other than IgG1 antibodies, the subclass antibody response was restricted to IgG3 and IgG4 antibodies in the acute and convalescent phases of infection, respectively. J. Med. Virol. 85:828–832, 2013. © 2013 Wiley Periodicals, Inc.     

Restoration of Glyoxalase Enzyme Activity Precludes Cognitive Dysfunction in a Mouse Model of Alzheimer’s Disease

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What is the Level of Evidence Substantiating Commercial Payers’ Coverage Policies for Total Joint Arthroplasty?

BackgroundThe prevalence of total joint arthroplasty (TJA) in the United States has drawn the attention of health care stakeholders. The payers have also used a variety of strategies to regulate the medical necessity of these procedures. The purpose of this study was to examine the level of evidence of the coverage policies being used by commercial payers in the United States.MethodsThe references of the coverage policies of four commercial insurance companies were reviewed for type of document, level of evidence, applicability to a TJA population, and success of nonoperative treatment in patients with severe degenerative joint disease.Results282 documents were reviewed. 45.8% were primary journal articles, 14.2% were level I or II, 41.2% were applicable to patients who were candidates for TJA, and 9.9% discussed the success of nonoperative treatment in patients who would be candidates for TJA.ConclusionMost of the references cited by commercial payers are of a lower level of scientific evidence and not applicable to patients considered to be candidates for TJA. This is relatively uniform across the reviewed payers. The dearth of high-quality literature cited by commercial payers reflects the lack of evidence and difficulty in conducting high level studies on the outcomes of nonoperative versus operative treatment for patients with severe, symptomatic osteoarthritis. Patients, surgeons, and payers would all benefit from such studies and we encourage professional societies to strive toward that end through multicenter collaboration.