Formation of frameshift-stimulating RNA pseudoknots is facilitated by remodeling of their folding intermediates.

Programmed -1 ribosomal frameshifting is an essential regulation mechanism of translation in viruses and bacteria. It is stimulated by mRNA structures inside the coding region. As the structure is unfolded repeatedly by consecutive translating ribosomes, whether it can refold properly each time is important in performing its function. By using single-molecule approaches and molecular dynamics simulations, we found that a frameshift-stimulating RNA pseudoknot folds sequentially through its upstream stem S1 and downstream stem S2. In this pathway, S2 folds from the downstream side and tends to be trapped in intermediates. By masking the last few nucleotides to mimic their gradual emergence from translating ribosomes, S2 can be directed to fold from the upstream region. The results show that the intermediates are greatly suppressed, suggesting that mRNA refolding may be modulated by ribosomes. Moreover, masking the first few nucleotides of S1 favors the folding from S2 and yields native pseudoknots, which are stable enough to retrieve the masked nucleotides. We hypothesize that translating ribosomes can remodel an intermediate mRNA structure into a stable conformation, which may in turn stimulate backward slippage of the ribosome. This supports an interactive model of ribosomal frameshifting and gives an insightful account addressing previous experimental observations.

Targeting cancer stemness mediated by BMI1 and MCL1 for non-small cell lung cancer treatment.

Lung cancer is the leading cause of cancer-associated death, with a global 5-year survival rate <20%. Early metastasis and recurrence remain major challenges for lung cancer treatment. The stemness property of cancer cells has been suggested to play a key role in cancer plasticity, metastasis and drug-resistance, and is a potential target for drug development. In this study, we found that in non-small cell lung cancer (NSCLC), BMI1 and MCL1 play crucial roles of cancer stemness including invasion, chemo-resistance and tumour initiation. JNK signalling serves as a link between oncogenic pathway or genotoxicity to cancer stemness. The activation of JNK, either by mutant EGFR or chemotherapy agent, stabilized BMI1 and MCL1 proteins through suppressing the expression of E3-ubiquitin ligase HUWE1. In lung cancer patient samples, high level of BMI1 is correlated with poor survival, and the expression of BMI1 is positively correlated with MCL1. A novel small-molecule, BI-44, was developed, which effectively suppressed BMI1/MCL1 expressions and inhibited tumour formation and progression in preclinical models. Targeting cancer stemness mediated by BMI1/MCL1 with BI-44 provides the basis for a new therapeutic approach in NSCLC treatment.

Coordination among tertiary base pairs results in an efficient frameshift-stimulating RNA pseudoknot.

Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes.

Targeting pathogenic macrophages by the application of SHP-1 agonists reduces inflammation and alleviates pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a progressive fibrotic disorder with no cure that is characterized by deterioration of lung function. Current FDA-approved drugs for IPF delay the decline in lung function, but neither reverse fibrosis nor significantly improve overall survival. SHP-1 deficiency results in hyperactive alveolar macrophages accumulating in the lung, which contribute to the induction of pulmonary fibrosis. Herein, we investigated whether employing a SHP-1 agonist ameliorates pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis murine model. Histological examination and micro-computed tomography images showed that SHP-1 agonist treatment alleviates bleomycin-induced pulmonary fibrosis. Reduced alveolar hemorrhage, lung inflammation, and collagen deposition, as well as enhanced alveolar space, lung capacity, and improved overall survival were observed in mice administered the SHP-1 agonist. The percentage of macrophages collected from bronchoalveolar lavage fluid and circulating monocytes in bleomycin-instilled mice were also significantly reduced by SHP-1 agonist treatment, suggesting that the SHP-1 agonist may alleviate pulmonary fibrosis by targeting macrophages and reshaping the immunofibrotic niche. In human monocyte-derived macrophages, SHP-1 agonist treatment downregulated CSF1R expression and inactivated STAT3/NFκB signaling, culminating in inhibited macrophage survival and perturbed macrophage polarization. The expression of pro-fibrotic markers (e.g., MRC1, CD200R1, and FN1) by IL4/IL13-induced M2 macrophages that rely on CSF1R signaling for their fate-determination was restricted by SHP-1 agonist treatment. While M2-derived medium promoted the expression of fibroblast-to-myofibroblast transition markers (e.g., ACTA2 and COL3A1), the application of SHP-1 agonist reversed the transition in a dose-dependent manner. Our report indicates that pharmacological activation of SHP-1 ameliorates pulmonary fibrosis via suppression of CSF1R signaling in macrophages, reduction of pathogenic macrophages, and the inhibition of fibroblast-to-myofibroblast transition. Our study thus identifies SHP-1 as a druggable target for the treatment of IPF, and suggests that the SHP-1 agonist may be developed as an anti-pulmonary fibrosis medication that both suppresses inflammation and restrains fibroblast-to-myofibroblast transition.

Empirical use of fluoroquinolones improves the survival of critically ill patients with tuberculosis mimicking severe pneumonia.

INTRODUCTION: Empirical use of fluoroquinolones may delay the initiation of appropriate therapy for tuberculosis (TB). This study aimed to evaluate the impact of empirical fluoroquinolone use on the survival of patients with pulmonary TB that mimicked severe community-acquired pneumonia (CAP) requiring intensive care. METHODS: Patients aged >18 years with culture-confirmed pulmonary TB who presented as severe CAP and were admitted to the ICU were divided into fluoroquinolone (FQ) and nonfluoroquinolone (non-FQ) groups based on the type of empirical antibiotics used. Those patients with previous anti-TB treatment or those who died within 3 days of hospitalization were excluded. The primary end point was 100-day survival. RESULTS: Of the 77 patients identified, 43 (56%) were in the FQ group and 34 (44%) were in the non-FQ group. The two groups had no statistically significant difference in co-morbidities (95% vs. 97%, P > 0.99) and Acute Physiology and Chronic Health Evaluation (APACHE) II scores (21.2 ± 7.1 vs. 22.5 ± 7.5, P = 0.46) on ICU admission. Overall, 91% and 82% of patients in the FQ and non-FQ groups, respectively, had sputum examinations for TB within 1 week of admission (P = 0.46), and results were positive in 7% and 15% (P = 0.47), respectively. For both groups, 29% received appropriate anti-TB therapy within 2 weeks after ICU admission. The 100-day mortality rate was 40% and 68% for the FQ and non-FQ groups, respectively (P = 0.02). By Cox regression analysis, APACHE score <20, no bacteremia during the ICU stay, and empirical fluoroquinolone use were independently associated with survival. CONCLUSION: Empirical use of fluoroquinolones may improve the survival of ICU patients admitted for pulmonary TB mimicking severe CAP.

Humoral Immunogenicity and Reactogenicity of the Standard ChAdOx1 nCoV-19 Vaccination in Taiwan.

BACKGROUND: The ChAdOx1 nCoV-19 vaccine has been widely administered against SARS-CoV-2 infection; however, data regarding its immunogenicity, reactogenicity, and potential differences in responses among Asian populations remain scarce. METHODS: 270 participants without prior COVID-19 were enrolled to receive ChAdOx1 nCoV-19 vaccination with a prime-boost interval of 8-9 weeks. Their specific SARS-CoV-2 antibodies, neutralizing antibody titers (NT50), platelet counts, and D-dimer levels were analyzed before and after vaccination. RESULTS: The seroconversion rates of anti-RBD and anti-spike IgG at day 28 after a boost vaccination (BD28) were 100% and 95.19%, respectively. Anti-RBD and anti-spike IgG levels were highly correlated (r = 0.7891), which were 172.9 ± 170.4 and 179.3 ± 76.88 BAU/mL at BD28, respectively. The geometric mean concentrations (GMCs) of NT50 for all participants increased to 132.9 IU/mL (95% CI 120.0-147.1) at BD28 and were highly correlated with anti-RBD and anti-spike IgG levels (r = 0.8248 and 0.7474, respectively). Body weight index was statistically significantly associated with anti-RBD IgG levels (p = 0.035), while female recipients had higher anti-spike IgG levels (p = 0.038). The GMCs of NT50 declined with age (p = 0.0163) and were significantly different across age groups (159.7 IU/mL for 20-29 years, 99.4 IU/mL for ≥50 years, p = 0.0026). Injection-site pain, fever, and fatigue were the major reactogenicity, which were more pronounced after prime vaccination and in younger participants (<50 years). Platelet counts decreased and D-dimer levels increased after vaccination but were not clinically relevant. No serious adverse events or deaths were observed. CONCLUSION: The vaccine is well-tolerated and elicited robust humoral immunity against SARS-CoV-2 after standard prime-boost vaccination in Taiwanese recipients.

TAZ negatively regulates the novel tumor suppressor ANKRD52 and promotes PAK1 dephosphorylation in lung adenocarcinomas.

Lung cancer is the leading cause of cancer death, and therefore the discovery of novel therapeutic targets is crucial. P21-activated kinase (PAK1) is an important oncogene involved in the signaling of actin cytoskeleton organization. Although PAK1 inhibition has been shown to suppress cancer progression, specific PAK1 inhibitors are not available due to the complex structure and insufficient understanding of this kinase. The Hippo signaling effector TAZ is known to be elevated in multiple human cancers and to promote cancer metastasis. This study aimed to explore the role of TAZ in regulating the tumor suppressor ankyrin repeat domain 52 (ANKRD52) and PAK1 activity. A negative correlation between TAZ and ANKRD52 was observed, with knockdown of TAZ leading to enhanced ANKRD52 promoter activity and increased mRNA levels. Moreover, reduced ANKRD52 levels were associated with late-stage lung cancer. Knockdowns of ANKRD52 resulted in elevated cell mobility, while forced ANKRD52 expression attenuated cell mobility. ANKRD52 is a subunit of the protein phosphatase 6 (PP6) holoenzyme. Mass spectrometry analysis revealed the interaction between PAK1 and the ANKRD52-PP6 complex. Knockdown of ANKRD52 or PP6c resulted in upregulated PAK1 phosphorylation. Our study demonstrates that the novel tumor suppressor protein ANKRD52 is transcriptionally inhibited by TAZ, regulating cell mobility through interactions with PP6c and dephosphorylation of PAK1.

P-Cresylsulfate, the Protein-Bound Uremic Toxin, Increased Endothelial Permeability Partly Mediated by Src-Induced Phosphorylation of VE-Cadherin.

The goal of our study was to investigate the impact of p-cresylsulfate (PCS) on the barrier integrity in human umbilical vein endothelial cell (HUVEC) monolayers and the renal artery of chronic kidney disease (CKD) patients. We measured changes in the transendothelial electrical resistance (TEER) of HUVEC monolayers treated with PCS (0.1-0.2 mM) similar to serum levels of CKD patients. A PCS dose (0.2 mM) significantly decreased TEER over a 48-h period. Both PCS doses (0.1 and 0.2 mM) significantly decreased TEER over a 72-h period. Inter-endothelial gaps were observed in HUVECs following 48 h of PCS treatment by immunofluorescence microscopy. We also determined whether PCS induced the phosphorylation of VE-cadherin at tyrosine 658 (Y658) mediated by the phosphorylation of Src. Phosphorylated VE-cadherin (Y658) and phosphorylated Src levels were significantly higher when the cells were treated with 0.1 and 0.2 mM PCS, respectively, compared to the controls. The endothelial barrier dysfunction in the arterial intima in CKD patients was evaluated by endothelial leakage of immunoglobulin G (IgG). Increased endothelial leakage of IgG was related to the declining kidney function in CKD patients. Increased endothelial permeability induced by uremic toxins, including PCS, suggests that uremic toxins induce endothelial barrier dysfunction in CKD patients and Src-mediated phosphorylation of VE-cadherin is involved in increased endothelial permeability induced by PCS exposure.

Hedgehog pathway maintains cell survival under stress conditions, and drives drug resistance in lung adenocarcinoma.

Hedgehog (HH) pathway plays an important role in embryonic development, but is largely inactive in adult except for tissue repair. Aberrant activation of HH pathway has been found in a variety of cancer types. In non-small cell lung cancer, however, the role and importance of HH pathway remain controversial. In the current study, we found that HH pathway was maintained in low activity in lung adenocarcinoma (LAC) cells under normal culture condition, but was highly induced in response to stress conditions. Activation of HH pathway promoted cell survival, growth, and invasion partially through HGF and MET signaling. Hedgehog-Interacting Protein (HHIP), a cell-surface negative regulator of HH pathway, was epigenetically silenced in LAC. Overexpression of HHIP blocked the activation of HH and HGF/MET pathways, and made cells significantly more susceptible to stress conditions. In LAC cells with acquired resistance to Epidermal Growth Factor Receptor Tyrosin Kinase Inhibitor (EGFR-TKI), we found that a part of tumor cells were much more sensitive to HH or HGF/MET inhibitors, suggesting an oncogenic addiction shift from EGFR to HH and HGF/MET pathways. In conclusion, this study showed that HH pathway is a survival signaling that drives LAC cell growth under stress conditions, and HHIP is a key regulator to block the induction of HH pathway. Targeting the HH pathway through inhibitors or HHIP thus holds promise to address EGFR-TKI resistance in LAC in clinic.

The intrinsic dynamics of Cse1p and Xpot elucidated by coarse-grained models.

Cse1p and Xpot are two karyopherin proteins that transport the corresponding cargos during the nucleocytoplasmic transport. We utilized Elastic Network Model (ENM) and Finite Element Analysis (FEA) to study their conformational dynamics. These dynamics were interpreted by their intrinsic modes that played key roles in the flexibility of karyopherins, which further affected the binding affinities. The findings included that it was the karyopherin's versatile conformations composed of the same superhelices of HEAT repeats that produced different degrees of functional flexibilities. We presented evidence that these coarse-grained methods could help to elucidate the biological function behind the structures of the two karyopherins.