Evaluating the efficacy of a rotating upper-room UVC-LED irradiation device in inactivating aerosolized Escherichia coli under different disinfection ranges, air mixing, and irradiation conditions.

Cost-effective and safe air disinfection methods are urgently needed in various environmental public settings. A novel UVC-based disinfection system was designed and tested to provide a promising solution because of its effective inactivation of indoor bioaerosols at a low cost. UVC light-emitting diodes (UVC-LEDs) were utilized as the irradiation source. This system has the unique feature of rotating the UVC-LEDs to generate a "scanning irradiation" zone. Escherichia coli was aerosolized into an experimental chamber, exposed to UVC-LEDs, and sampled using an impactor. Effects of air mixing (well-mixed vs. poorly-mixed), transmission range (short vs. long), and irradiation mode (stationary vs. rotating) were evaluated. The system performs significantly well under the poorly-mixed condition. The results obtained from the short disinfection range indicate that the rotating UVC was approximately 70.5 % more effective than the stationary UVC for the poorly-mixed case. Further, we evaluated the performance of the long disinfection range under a poorly-mixed situation, and the disinfection efficacy was 84.6 % higher for the rotating irradiation than that of the stationary. About 0.59-1.34 J/m2 UV dose can be used to obtain one-log inactivation of E. coli. In conclusion, the novel rotating upper-room UVC-LED system is effective in reducing indoor pathogen transmission, and our findings are highly significant to a growing field where LEDs are applied for disinfection.

Study of synergistic disinfection by UVC and positive/negative air ions for aerosolized Escherichia coli, Salmonella typhimurium, and Staphylococcus epidermidis in ventilation duct flow.

The efficacy of the in-duct application of ultraviolet waveband C (UVC) emitting at 254 nm wavelength and air ions against aerosolized bacteria was studied in a full-scale 9-m long ventilation duct. Combined positive and negative ion polarities (bipolar ions) and combined UVC and ions were tested. The UVC was generated by a mercury-type UVC lamp and air ions were generated by positive and negative polarity ionizers. Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium), and Staphylococcus epidermidis (S. epidermidis)were tested at a concentration of 108 to 109 cells in 50 ml of sterilized distilled water. The case in which the positive ionizer was placed first, followed by the negative ionizer, demonstrated significantly higher disinfection efficiencies for E. coli (p = 0.007) and S. typhimurium (p < 0.001), but lower efficiency for S. epidermidis (p = 0.01) than the reversed sequence. The combination of UVC (3.71 J/m2 ) and air ions (1.13 × 1012  ions/m3 for positive ions and 8.00 × 1011  ions/m3 for negative ions) led to higher inactivation than individual disinfection agents operating under the same dose. A synergetic inactivation effect was observed for S. epidermidis under the combined UVC and positive ion case, while the combined UVC and negative ion case showed significant synergy effects for E. coli and S. typhimurium.

A novel upper-room UVC-LED irradiation system for disinfection of indoor bioaerosols under different operating and airflow conditions.

The potential of inactivating indoor bacteria aerosols using a novel rotating ultraviolet-C (UV-C) light-emitting-diode (LED) system was investigated. The system was installed in the upper level of a full scale chamber and its effectiveness against aerosolized E. coli, S. marcescens, and S. epidermidis under the well-mixed with stationary UV-LED scenario was initially tested. The estimated susceptibility values were 1.068, 1.148, and 0.156 m2/J for E. coli, S. marcescens, and S. epidermidis, respectively. Three additional scenarios of experiments were conducted, in which E. coli was aerosolized into the test chamber and then allowed to decay under (i) poorly-mixed condition with stationary system, (ii) well-mixed with rotating system, and (iii) poorly-mixed conditions with rotating system. Our results showed no significant difference between the performance of stationary and rotating UR-UVGI-LED systems under a well-mixed condition. While the performance of the stationary UR-UVGI-LED system under a poorly-mixed condition decreased by 52.90-79.38 % compared to a well-mixed condition, rotating the UR-UVGI-LED system under a poorly-mixed condition, compared to the stationary system, enhanced its performance by 22.36-49.86 %. Thus, our proposed rotating irradiation offers great potential for application in environments where bioaerosols are unevenly distributed in a built environment.

Chronic activation of mTOR complex 1 is sufficient to cause hepatocellular carcinoma in mice.

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a nutrient-sensitive protein kinase that is aberrantly activated in many human cancers. Whether dysregulation of mTORC1 signaling in normal tissues increases the risk for cancer, however, is unknown. We focused on hepatocellular carcinoma, which has been linked to environmental factors that affect mTORC1 activity, including diet. Ablation of the gene encoding TSC1 (tuberous sclerosis complex 1), which as part of the TSC1-TSC2 complex is an upstream inhibitor of mTORC1, results in constitutively increased mTORC1 signaling, an effect on this pathway similar to that of obesity. We found that mice with liver-specific knockout of Tsc1 developed sporadic hepatocellular carcinoma with heterogeneous histological and biochemical features. The spontaneous development of hepatocellular carcinoma in this mouse model was preceded by a series of pathological changes that accompany the primary etiologies of this cancer in humans, including liver damage, inflammation, necrosis, and regeneration. Chronic mTORC1 signaling led to unresolved endoplasmic reticulum stress and defects in autophagy, factors that contributed to hepatocyte damage and hepatocellular carcinoma development. Therefore, we conclude that increased activation of mTORC1 can promote carcinogenesis and may thus represent a key molecular link between cancer risk and environmental factors, such as diet.

Akt stimulates hepatic SREBP1c and lipogenesis through parallel mTORC1-dependent and independent pathways.

Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2.

Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway.

BACKGROUND: The signaling pathways imposing hormonal control over adipocyte differentiation are poorly understood. While insulin and Akt signaling have been found previously to be essential for adipogenesis, the relative importance of their many downstream branches have not been defined. One direct substrate that is inhibited by Akt-mediated phosphorylation is the tuberous sclerosis complex 2 (TSC2) protein, which associates with TSC1 and acts as a critical negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Loss of function of the TSC1-TSC2 complex results in constitutive mTORC1 signaling and, through mTORC1-dependent feedback mechanisms and loss of mTORC2 activity, leads to a concomitant block of Akt signaling to its other downstream targets. METHODOLOGY/PRINCIPAL FINDINGS: We find that, despite severe insulin resistance and the absence of Akt signaling, TSC2-deficient mouse embryo fibroblasts and 3T3-L1 pre-adipocytes display enhanced adipocyte differentiation that is dependent on the elevated mTORC1 activity in these cells. Activation of mTORC1 causes a robust increase in the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master transcriptional regulator of adipocyte differentiation. In examining the requirements for different Akt-mediated phosphorylation sites on TSC2, we find that only TSC2 mutants lacking all five previously identified Akt sites fully block insulin-stimulated mTORC1 signaling in reconstituted Tsc2 null cells, and this mutant also inhibits adipogenesis. Finally, renal angiomyolipomas from patients with tuberous sclerosis complex contain both adipose and smooth muscle-like components with activated mTORC1 signaling and elevated PPARgamma expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that activation of mTORC1 signaling is a critical step in adipocyte differentiation and identifies TSC2 as a primary target of Akt driving this process. Therefore, the TSC1-TSC2 complex regulates the differentiation of mesenchymal cell lineages, at least in part, through its control of mTORC1 activity and PPARgamma expression.

S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt.

Feedback inhibition of the PI3K-Akt pathway by the mammalian target of rapamycin complex 1 (mTORC1) has emerged as an important signaling event in tumor syndromes, cancer, and insulin resistance. Cells lacking the tuberous sclerosis complex (TSC) gene products are a model for this feedback regulation. We find that, despite Akt attenuation, the Akt substrate GSK3 is constitutively phosphorylated in cells and tumors lacking TSC1 or TSC2. In these settings, GSK3 phosphorylation is sensitive to mTORC1 inhibition by rapamycin or amino acid withdrawal, and GSK3 becomes a direct target of S6K1. This aberrant phosphorylation leads to decreased GSK3 activity and phosphorylation of downstream substrates and contributes to the growth-factor-independent proliferation of TSC-deficient cells. We find that GSK3 can also be regulated downstream of mTORC1 in a HepG2 model of cellular insulin resistance. Therefore, we define conditions in which S6K1, rather than Akt, is the predominant GSK3 regulatory kinase.

Lipase-selective functional domains of perilipin A differentially regulate constitutive and protein kinase A-stimulated lipolysis.

Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1-300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301-517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1-300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301-517 (Ser433, Ser492, and Ser517). Peri 301-517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301-517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.

Tumor necrosis factor-alpha stimulates lipolysis in differentiated human adipocytes through activation of extracellular signal-related kinase and elevation of intracellular cAMP.

Tumor necrosis factor-alpha (TNF-alpha) stimulates lipolysis in human adipocytes. However, the mechanisms regulating this process are largely unknown. We demonstrate that TNF-alpha increases lipolysis in differentiated human adipocytes by activation of mitogen-activated protein kinase kinase (MEK), extracellular signal-related kinase (ERK), and elevation of intracellular cAMP. TNF-alpha activated ERK and increased lipolysis; these effects were inhibited by two specific MEK inhibitors, PD98059 and U0126. TNF-alpha treatment caused an electrophoretic shift of perilipin from 65 to 67 kDa, consistent with perilipin hyperphosphorylation by activated cAMP-dependent protein kinase A (PKA). Coincubation with TNF-alpha and MEK inhibitors caused perilipin to migrate as a single 65-kDa band. Consistent with the hypothesis that TNF-alpha induces perilipin hyperphosphorylation by activating PKA, TNF-alpha increased intracellular cAMP approximately 1.7-fold, and the increase was abrogated by PD98059. Furthermore, H89, a specific PKA inhibitor, blocked TNF-alpha-induced lipolysis and the electrophoretic shift of perilipin, suggesting a role for PKA in TNF-alpha-induced lipolysis. Finally, TNF-alpha decreased the expression of cyclic-nucleotide phosphodiesterase 3B (PDE3B) by approximately 50%, delineating a mechanism by which TNF-alpha could increase intracellular cAMP. Cotreatment with PD98059 restored PDE3B expression. These studies suggest that in human adipocytes, TNF-alpha stimulates lipolysis through activation of MEK-ERK and subsequent increase in intracellular cAMP.