Ubiquitination and regulation of Smad7 in the TGF-ß1/Smad signaling of aristolochic acid nephropathy.

Aristolochic acid I (AAI) affects TGF-ß1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-ß1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10 µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TßRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TßRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-ß1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-ß1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TßRI.

Assessment the protection performance of different level personal respiratory protection masks against viral aerosol.

New viral disease such as SARS and H1N1 highlighted the vulnerability of healthcare workers to aerosol-transmitted viral infections. This paper was to assess the protection performance of different level personal respiratory protection equipments against viral aerosol. Surgical masks, N95 masks and N99 masks were purchased from the market. The masks were sealed onto the manikin in the aerosol testing chamber. Viral aerosol was generated and then sampled simultaneously before and after the tested mask using biosamplers. This allows a percentage efficiency value to be calculated against test phage SM702 aerosols which surrogates of viral pathogens aerosol. At the same time, the masks face fit factor was determined by TSI8020. The viral aerosol particles aerodynamic diameter was 0.744 µm, and GSD was 1.29. The protection performance of the material of all the tested masks against viral aerosol was all >95 %. All the five surgical masks face fit factor were <8. F model N95 mask and H model N99 mask face fit factor were all >160. G model N95 mask face fit factor was 8.2. The protection performances of N95 or N99 masks were many times higher than surgical mask when considering the face fit factor. Surgical masks cannot offer sufficient protection against the inhalation of viral aerosol because they cannot provide a close face seal.

Impact of relative humidity and collection media on mycobacteriophage D29 aerosol.

This study was conducted to evaluate the effect of aerosol generation, methods of sampling, storage conditions, and relative humidity on the culturability of the mycobacteriophage D29. The lytic phage D29 can kill Mycobacterium tuberculosis, and the phage aerosol can be treated as a potential tool for tuberculosis treatment. The culturability of D29 was tested using a test chamber designed for the bioaerosols research against three spray liquids (deionized water, phosphate-buffered saline [PBS], and normal saline), four collection media (suspension medium [SM], nutrient broth, PBS, and deionized water), two sampling systems (the all-glass impinger AGI-30 and the Biosampler) and across a range of humidities (20 to 90%). The effect of storage conditions on the culturability of collected sample was also evaluated for the AGI-30 impinger. The results proved that viable phage D29 particles generated by deionized water were approximately 30- and 300-fold higher than PBS and normal saline, respectively. As collection media, SM buffer and nutrient broth were observed to yield a higher number of plaques compared to PBS and deionized water. No difference was observed in collection efficiency between AGI-30 and Biosampler with two detection methods (culture-based technique and real-time PCR). The culturability of collected D29 in SM buffer or nutrient broth can be maintained up to 12 h irrespective of storage temperature. Relative humidity was found to strongly influence airborne D29 culturability which is 2- to 20-fold higher in low humidity (25%) than medium (55%) or high (85%) humidity. This research will help identify the optimal means for the application of D29 aerosol in animal inhalation experiments.

[Evaluation of the protective performance of a positive pressure bio-protective clothing against viral aerosol].

OBJECTIVE: To evaluate the protective performance of a positive pressure bio-protective clothing against viral aerosol. METHODS: The suspension of indicating virus phage Phi-X174 was made for viral aerosol generating in a hermetic cabin. The diameter of viral aerosol particles were measured with a aerodynamics size analyzer. By adjusting the inner humidity of the cabin, the protective efficiency of the positive pressure bio-protective clothing against viral aerosol in high and low windshield conditions was determined with Andersen six-stage air sampler sampling and plage forming unit (PFU) counting, respectively. RESULTS: The mass median diameter of Phage Phi-X174 aerosol particles was about 0.922 µm and the background concentration is beyond 2 × 104 particles/m³. The protective efficiency of the clothing against phage Phi-X174 aerosol particles was above 99.9% under different test conditions with the range of viral aerosol concentration between 0 - 23 PFU/m³. Airflow (P = 0.84), environment humidity conditions (P = 0.33) and sampling time (P = 0.07) did not affect the protective efficiency statistically. CONCLUSION: The positive pressure bio-protective clothing provided a relatively high efficiency against phage Phi-X174 aerosol regardless of airflow rate, environment humidity and sampling time.

Inhalation Study of Mycobacteriophage D29 Aerosol for Mice by Endotracheal Route and Nose-Only Exposure.

BACKGROUND: Lytic mycobacteriophage D29 has the potential for tuberculosis treatment including multidrug-resistant strains. The aims of this study are to investigate deposition and distribution of aerosolized phage D29 particles in naive Balb/C mice, together with pharmacokinetics and evaluation of acute lung injury. METHODS: Pharmacokinetics and BALF (bronchoalveolar lavage fluids) were analyzed after administration of phage D29 aerosols by endotracheal route using Penn-century aerosolizer; Collison 6-jet and Spinning top aerosol nebulizers (STAG) were used to generate phage aerosols with different particle size distributions in nose-only inhalation experiments. After exposure, deposited amounts of phage D29 particles in respiratory tracts were measured, and deposition efficiencies were calculated. A typical path deposition model for mice was developed, and then comparisons were made between predictions and experimentally measured results. RESULTS: Approximately 10% of aerosolized phages D29 reached lung of mouse for pulmonary delivery, and were completely eliminated until 72 h after administration. In contrast, about 0.1% of intraperitoneal injected phages reached the lung, and were almost eliminated at 12 h time point. The inflammation was hardly observed in lung according to the results of BALF analysis. The CMADs (count median aerodynamic diameters) of generated aerosol by Collison and STAG nebulizer were 0.8 µm and 1.5 µm, respectively. After nose-only exposure, measured deposition efficiencies in whole respiratory tract for 0.8 and 1.5 µm phage particles were below 1% and 10%, respectively. Predictions of the computer deposition model compared fairly well with experimentally measured results. CONCLUSIONS: This is the first systematic study of phage D29 aerosol respiratory challenge in laboratory animals. It provides evidence that aerosol delivery of phage D29 is an effective way for treating pulmonary infections caused by Mycobacterium tuberculosis. This research will also provide important data for future inhalation experiments.

Assessment of the risk of infectious aerosols leaking to the environment from BSL-3 laboratory HEPA air filtration systems using model bacterial aerosols.

To assess the risk of infectious bacterial aerosols leaking to the environment, the filtration efficiency of a biosafety level 3 (BSL-3) laboratory high-efficiency particulate (HEPA) filter was investigated using the aerosolized bacteria Serratia marcescens. The aerosol size was measured using an Andersen sampler. Eight first stage HEPA filters (numbered 1-8) were distributed in contaminated labs and exhausts from each of the first stage HEPA filters were aggregated and filtered through one second stage HEPA filter before being released to the environment. In total, 8 first-stage and 1 second-stage HEPA filters from the BSL-3 air purification system were analyzed. No S. marcescens was detected in first stage filters 1, 2, 4, 5, 7 and 8 and the second stage HEPA filter. The filtration efficiencies against aerosolized S. marcescens were >99.9999%. First stage filter numbers 3 and 6 had filtration efficiencies of 99.9825% and 99.9906%, respectively. When filter number 3 was replaced by a new filter and the bracket for filter number 6 was sealed, no aerosolized S. marcescens was detected in the filtered air. Our work suggests that the BSL-3 laboratory HEPA filter air purification system is effective against bacterial aerosols, with little to no bacterial leakage into the environment.

Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column.