RESUMO
Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first- and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)-emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CS-induced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS-exposed murine model and human COPD-emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P < 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P < 0.001) colocalize with autophagy protein microtubule-associated protein 1 light chain-3B(+) autophagy bodies, whereas U.S. Food and Drug Administration-approved autophagy-inducing drug (carbamazepine) significantly (P < 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein-p62 protein-protein interaction (P < 0.0005) and p62 expression (aberrant autophagy marker; P < 0.0001), verifying CS-impaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P < 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P < 0.001), confirming involvement of aggresome bodies in COPD-emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD-emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD-emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD-emphysema pathogenesis.
Assuntos
Agregação Patológica de Proteínas/metabolismo , Enfisema Pulmonar/metabolismo , Fumar/efeitos adversos , Proteínas Ubiquitinadas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Autofagia , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Agregação Patológica de Proteínas/etiologia , Agregação Patológica de Proteínas/patologia , Transporte Proteico , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , Proteínas de Ligação a RNA/metabolismo , Proteína com ValosinaRESUMO
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-µm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.
Assuntos
Dissecação/métodos , Fixadores/química , Formaldeído/química , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Inclusão em Parafina/métodos , Fixação de Tecidos/métodos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Confiabilidade dos Dados , Receptores ErbB/genética , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Pulmão , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Oncologia/métodos , Mutação , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos TestesRESUMO
We evaluated the microbiological performance of an ultraviolet (UV) light-based peritoneal dialysis catheter connection system. The system includes a UV light-generating device combined with a UV transmissive window incorporated into the transfer set. Each UV transparent transfer set was inoculated with 10 µL of cultured inoculum consisting of either S. aureus, E. coli, or C. albicans After being inoculated, we attached a solution set connector to the transfer catheter, and exposed that connection to a UV light dose of approximately 340 mJoules/cm2 After exposure to UV light, we broke the seal of the solution set and opened the plunger valve on the UV transmissive transfer catheter. We then flushed 10 mL of dialysate through the connection. The flushed solution was collected, diluted, plated on agar medium, and incubated for 24 hours. Results were compared to positive controls collected in an identical manner without exposure to UV light. Thirty test samples and 3 positive controls were collected for each organism. All test samples exposed to UV light had complete kill of bacteria except 1 colony on a single plate in the S. aureus group. Mean log reduction was 4.03 for C. albicans, 4.73 for S. aureus, and 5.29 for E. coli All positive control samples had significant bacterial growth. Our results demonstrate that the application of UV light within a UV transmissive transfer catheter window produces a germicidal effect upon microorganisms known to be associated with peritonitis.