Development of a Rapid Epstein-Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay.

The quality of cellular products used in biological research can directly impact the ability to obtain accurate results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. We developed three EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 103 copy numbers for the RPA-based and RPA-LFA systems and 1 × 104 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those obtained by the EBV assay methods specified in the pharmaceutical industry standards of the People's Republic of China. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

How active social network site use affects green consumption: A moderated mediation model.

A growing body of literature suggests a link between the usage of social networking sites (SNSs) and green consumption. However, researchers have shown that not all types of SNS usage have the same effect on individuals; therefore, to fully understand the relationship between a particular SNS use type and green consumption, as well as the mechanisms underlying the relationship, more research is required. This study examined a moderated mediation model based on self-awareness theory to explain the "how" and "why" of the relationship between active SNS use and green consumption. An offline survey (N = 210) and an online survey (N = 348) were conducted. The results suggest that active SNS use is positively associated with green consumption via public self-awareness and that impression management motives moderate the mediating role of public self-awareness in the relationship between active SNS use and green consumption. By examining the connection between a specific type of SNS use (active SNS use) and green consumption, our study adds to the body of literature on the causes of green consumption. The results have substantial implications for future research promoting socially responsible consumption behavior.

Ubiquitination-Related miRNA-mRNA Interaction Is a Potential Mechanism in the Progression of Retinoblastoma.

Purpose: Retinoblastoma (RB) is the most common primary malignant intraocular cancer. The etiology of RB is complex, and the mechanisms driving its progression remain unclear. Here, we used a series of bioinformatics approaches and experimental methods to investigate the potential regulatory mechanism involved in RB progression. Methods: The common differentially expressed genes were obtained from the public dataset GSE97508. Protein-protein interaction (PPI) network, correlation, and functional enrichment analyses were carried out. The candidate genes were verified in different RB cell lines, and ARPE19 cells served as control. miRNA-mRNA interaction analysis was performed and confirmed by real-time PCR. The CCK-8 assay was conducted to detect cell viability, and the transwell assay was utilized for evaluating the abilities of cell migration and invasion. Results: Overall, a total of 258 common differentially expressed genes associated with RB progression were screened out. The PPI network analysis further identified eight downregulated genes mainly enriched in the protein ubiquitination pathway. Moreover, we confirmed UBE2E1, SKP1, FBXO9, FBXO15, and RNF14 from among eight genes through experimental validation in vitro. Furthermore, miRNA-mRNA interaction and real-time PCR analysis of five hub genes revealed that ubiquitination-related miR-548k was involved in RB progression. Loss- and gain-of-function experiments demonstrated that miR-548k and its targets were essential for cell viability, migration, and invasion in the RB cells. Conclusions: Our data indicate that the dysregulation of protein ubiquitination may play an important role in RB progression, and ubiquitination-related miR-548k may be a promising therapeutic target for RB.

Secondary Structure in Nonpeptidic Supramolecular Block Copolymers.

Proteins contain a level of complexity-secondary and tertiary structures-that polymer chemists aim to imitate. The bottom-up synthesis of protein-mimicking polymers mastering sequence variability and dispersity remains challenging. Incorporating polymers with predefined secondary structures, such as helices and π-π stacking sheets, into block copolymers circumvents the issue of designing and predicting one facet of their 3D architecture. Block copolymers with well-defined secondary-structure elements formed by covalent chain extension or supramolecular self-assembly may be considered for localized tertiary structures.In this Account, we describe a strategy toward block copolymers composed of units bearing well-defined secondary structures mixed in a "plug-and-play" manner that approaches a modicum of the versatility seen in nature. Our early efforts focused on the concept of single-chain collapse to achieve folded secondary structures through either hydrogen bonding or quadrupole attractive forces. These cases, however, required high dilution. Therefore, we turned to the ring-opening metathesis polymerization (ROMP) of [2.2]paracyclophane-1,9-dienes (pCpd), which forms conjugated, fluorescent poly(p-phenylenevinylene)s (PPVs) evocative of ß-sheets. Helical building blocks arise from polymers such as poly(isocyanide)s (PICs) or poly(methacrylamide)s (PMAcs) containing bulky, chiral side groups while the coil motif can be represented by any flexible chain; we frequently chose poly(styrene) (PS) or poly(norbornene) (PNB). We installed moieties for supramolecular assembly at the chain ends of our "sheets" to combine them with complementary helical or coil-shaped polymeric building blocks.Assembling hierarchical materials tantamount to the complexity of proteins requires directional interactions with high specificity, covalent chain extension, or a combination of both chemistries. Our design is based on functionalized reversible addition-fragmentation chain-transfer (RAFT) agents that allowed for the introduction of recognition motifs at the terminus of building blocks and chain-terminating agents (CTAs) that enabled the macroinitiation of helical polymers from the chain end of ROMP-generated sheets and/or coils. To achieve triblock copolymers with a heterotelechelic helix, we relied on supramolecular assembly with helix and coil-shaped building blocks. Our most diverse structures to date comprised a middle block of PPV sheets, parallel or antiparallel, and supramolecularly or covalently linked, respectively, end-functionalized with molecular recognition units (MRUs) for orthogonal supramolecular assembly. We explored PPV sheets with multiple folds achieved by chain extension using alternating pCpd and phenyl-pentafluorophenyl ß-hairpin turns. Using single-molecule polarization spectroscopy, we showed that folding occurs preferentially in multistranded over double-stranded PPV sheets. Our strategy toward protein-mimicking and foldable polymers demonstrates an efficient route toward higher ordered, well-characterized materials by taking advantage of polymers that naturally manifest secondary structures. Our studies demonstrate the retention of distinct architectures after complex assembly, a paradigm that we believe may extend to other polymeric folding systems.

A novel chemosensor for the distinguishable detections of Cu2+ and Hg2+ by off-on fluorescence and ratiometric UV-visible absorption.

A novel dual-functional chemosensor, derived from the conjugation of rhodamine B with a quinoline derivative (RHQ), was firstly synthesized with high efficiency and cost-effectiveness for the distinguishable detections of Cu2+ and Hg2+ via ring-opening and ring-forming mechanism. The chemosensor exhibits highly selective and distinguishable responses for Cu2+ and Hg2+ in CH3CN-H2O (4:1, v/v) with off-on fluorescence and ratiometric ultraviolet-visible (UV-Vis) absorption changes. Additionally, Cu2+ is identified by opening a rhodamine spirocycle with a UV-Vis absorption band, at around 560 nm and fluorescence turn-on. Interestingly, Hg2+ is discerned by opening the rhodamine spirocycle and by generating a new special cycle for the quinoline unit. Resultantly, there were two UV-Vis absorption bands at around 365 nm and 560 nm, which were accompanied by fluorescence turn-on. Moreover, the chemosensor can quantitatively detect Cu2+ and Hg2+ by off-on fluorescence and ratiometric UV-Vis absorption changes, respectively. Furthermore, the chemosensor with low cytotoxicity could be successfully administered to monitor Cu2+ and Hg2+ in living cells. This work may pay the way for the development of dual-functional chemosensor for quantificationally detecting metal ions in environmental and biological systems.

A dual-channel chemosensor based on 8-hydroxyquinoline for fluorescent detection of Hg2+ and colorimetric recognition of Cu2.

A novel dual-channel chemosensor, 7-allylquinolin-8-ol (AQ), was synthesized based on 8-hydroxyquinoline for selective fluorescence detection of Hg2+ and colorimetric recognition of Cu2+. The chemosensor reacted with Hg2+ and generated a new Hg-containing compound with significantly enhanced fluorescence, which turned from faint blue to strong green. Further experiments indicated that AQ could be used to quantitatively detect Hg2+ via fluorescence spectroscopy with a low detection limit (2.1 nM). The good reversibility of the synthesized chemosensor was also demonstrated using NaBH4. Moreover, AQ was successfully used for the detection of Cu2+ through the formation of a stable coordination compound, which exhibited an ultraviolet-visible (UV-Vis) ratiometric change, while its color changed from colorless to pale yellow under natural light. Additional experiments using various Cu2+ concentrations showed that the developed chemosensor could be further employed for the quantitative ratiometric estimation of Cu2+ by UV-Vis.

Role of Cigarette Smoke-Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis.

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.

Critical role of VCP/p97 in the pathogenesis and progression of non-small cell lung carcinoma.

BACKGROUND: Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05). CONCLUSIONS/SIGNIFICANCE: Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.