We have previously shown that the G protein ofvesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracyclinedependent and can be modulated by 18-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.Retroviral vectors derived from Moloney murine leukemia virus have been used extensively to transduce genes into mammalian cells (1, 2). However, problems such as the inability to infect quiescent cells, low virus titers, and relatively poor infectivity in human cells severely limit the application of retroviral vectors in gene transfer. To overcome some of these problems, we have generated retroviral vectors pseudotyped with the G protein of vesicular stomatitis virus (VSV-G) (3-5). We have shown that the pseudotyped retroviral vectors have an expanded host range and are able to infect many cell types with a greater efficiency than traditional amphotropic retroviral vectors (3-6). Moreover, the pseudotyped virus can be concentrated by ultracentrifugation to titers greater than 109 colony forming units/ml (4). These unique properties of the VSV-G pseudotyped virus not only extend the use of retroviral vectors for genetic studies in previously inaccessible species, but also facilitate more efficient preclinical and clinical studies of the potential for human gene therapy.However, the generation of stable packaging cell lines for pseudotyped retroviral vectors has been difficult because of the toxicity of VSV-G in most mammalian cells (4, 5). To overcome this problem, we have modified the tetracyclineinducible system (7) to regulate the expression of VSV-G. In that system, a chimeric transcription factor, tTA, was produced by combining the repressor of Escherichia coli tetracyclineresistance operon (tetR) and the activation domain of virion protein 16 (VP16) of herpes simplex virus (HSV). This chimeric transactivator can regulate gene expression from a minimal promoter linked to multiple copies of tetO, the binding site for the tetracycline repressor. In the absence of tetracycline, tTA binds the tetO site and activates transcription from the tetO-containing promoter. In the presence of tetracycline, interaction of tTA with the tetO is prevented by its binding to tetracycline, thereby prevents transcription.While the expression of several genes has been reported to be inducible using this system (7-11), establishment of cell lines stably expressing tTA has been difficult, presumably because the activation domain of VP16 is capable of squelching general cellular transcription when expressed in l...

Fasting is known to have many health benefits such as prolonging lifespan and suppression of tumorigenesis. [1][2][3] In the present study, we systematically evaluated the effects of water-only fasting on metabolic-syndrome and age-related risk markers in 45 normal-weight individuals.As shown, a 4.59 kg reduction in body weight, 9.85 cm reduction in waist circumference, and 1.64 kg/m 2 reduction in body mass index (BMI) were observed during a 5-day water-only fast (Figures 1A-1C). After refeeding for 1 month (day 38), body weight, waist circumference, and BMI were still lower than the baseline level . Blood pressure (BP) significantly declined during water-only fasting with diastolic BP declining more than systolic BP and gradually both increased to the baseline level by 98 d (Figures 1D and 1E). Considering many fasting studies showed diastolic BP reduction did not exceed systolic BP reduction, future studies are needed on water-only fasting and BP reduction. Insulin dropped approximately 2.8-fold lower than the baseline level during water-only fasting (Figure 1F). Insulin-like growth factor 1 (IGF-1) decreased by a total of 26% during water-only fasting and decreased more in females than males (Figure 1G and Table S1). Future studies will address the sexual disparity of IGF-1 reduction during water-only fasting. The number of pan T cells, CD4+T cells, CD8+T cells, and B cells decreased during water-only fasting . In contrast, the frequency of Treg cells significantly increased during fasting and still exceeded the baseline level 3 months after refeeding (Figures 1L and 1M). This is an important benefit, since Treg cells have anti-inflammation effects. 4 With regard to thyroid hormones, T4 increased rapidly during fasting, whereas T3 and TSH decreased (Figures 1N-1P). The decreased level of T3 during water-only fasting is of particularly importance since a low T3 level, without impairing thyroid function, is strongly associated with longevity. 5,6 The present studyThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

We examined the role of Tollip in the hypertrophic response of cardiomyocytes. C57BL/6 mice were subjected to transverse aortic constriction (TAC) for 2 weeks and age-matched sham surgical operated mice served as control. TAC significantly reduced the association of Tollip with IRAK-1 by 66.4 percent and increased NF-kappaB binding activity by 86.5 percent and the levels of phospho-p38 by 114.6 percent in the myocardium compared with sham control, respectively. In vitro experiments showed that IL-1beta stimulation also significantly reduced the association of Tollip with IRAK-1 and increased NF-kappaB binding activity in neonatal cardiomyocytes. Tollip overexpression by transfection of cardiac myocytes significantly attenuated the IL-1beta-induced hypertrophic response of cardiac myocytes as evidenced by reduced cell size (16.4 percent) and decreased ANP expression (33.3 percent). Overexpression of Tollip also reduced NF-kappaB binding activity by 30.7 percent and phospho-p38 by 47.1 percent, respectively. The results suggest that Tollip could be a negative regulator during the development of cardiac hypertrophy. The negative regulation of cardiac hypertrophy by Tollip may involve downregulation of the MyD88-dependent NF-kappaB activation pathway.

The present study aimed to explore genetic association of receptor activator nuclear factor κB (RANK) polymorphisms with individual susceptibility to knee osteoarthritis (OA) in different Kellgren–Lawrence (KL) grades. This case–control study included 138 knee OA patients and 145 healthy individuals. RANK rs1805034 and rs8086340 polymorphisms were genotyped through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The effects of RANK polymorphisms on knee OA risk were analyzed via χ 2 test or Fisher exact test, and the results were expressed using odds ratios (ORs) with corresponding 95% confidence intervals (CIs). The C allele of rs1805034 polymorphism had significantly higher frequency in knee OA patients than in controls ( P = .044), indicating that this allele could increase the risk of knee OA (OR = 1.424, 95% CI = 1.010–2.008). Besides, the CC genotype and C allele of the rs1805034 polymorphism were significantly associated with elevated risk of knee OA in moderate grade (CC vs TT: P = .018, OR = 3.071, 95% CI = 1.187–7.941; C vs T: P = .012, OR = 1.787, 95% CI = 1.131–2.823). However, rs8086340 polymorphism had no significant association with knee OA risk The C allele of RANK rs1805034 polymorphism is closely correlated with increased risk of knee OA, especially for moderate grade.

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