Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice.

BACKGROUND: Recent advances in gene editing technology have opened up new avenues for in vivo gene therapy, which holds great promise as a potential treatment method for dilated cardiomyopathy (DCM). The CRISPR-Cas13 system has been shown to be an effective tool for knocking down RNA expression in mammalian cells. PspCas13b, a type VI-B effector that can be packed into adeno-associated viruses and improve RNA knockdown efficiency, is a potential treatment for diseases characterized by abnormal gene expression. RESULTS: Using PspCas13b, we were able to efficiently and specifically knockdown the mutant transcripts in the AC16 cell line carrying the heterozygous human TNNT2R141W (hTNNT2R141W) mutation. We used adeno-associated virus vector serotype 9 to deliver PspCas13b with specific single guide RNA into the hTNNT2R141W transgenic DCM mouse model, effectively knocking down hTNNT2R141W transcript expression. PspCas13b-mediated knockdown significantly increased myofilament sensitivity to Ca2+, improved cardiac function, and reduced myocardial fibrosis in hTNNT2R141W DCM mice. CONCLUSIONS: These findings suggest that targeting genes through Cas13b is a promising approach for in vivo gene therapy for genetic diseases caused by aberrant gene expression. Our study provides further evidence of Cas13b's application in genetic disease therapy and paves the way for future applicability of genetic therapies for cardiomyopathy.

Rational design of small-molecules to recognize G-quadruplexes of c-MYC promoter and telomere and the evaluation of their in vivo antitumor activity against breast cancer.

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand-G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.

LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole.

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110-CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110-CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.

Chylothorax following posterior low lumbar fusion surgery: A case report.

BACKGROUND: Postoperative chylothorax is usually regarded as a complication associated with cardiothoracic surgery; however, it is one of the rare complications in orthopedic surgery. This case report describes a female patient who developed chylothorax after a successful L4-S1 transforaminal lumbar interbody fusion surgery. The etiology, diagnosis, and treatment were analyzed and discussed. CASE SUMMARY: A 50-year-old woman was admitted with repeated back and leg pain. She was diagnosed with L4 degenerative spondylolisthesis, L4/L5 and L5/S1 intervertebral disc herniation and L5 instability, and underwent successful posterior L4-S1 instrumentation and fusion surgery. Unfortunately, thoracic effusion was identified 2 d after operation. The thoracic effusion was finally confirmed to be chylous based on twice positive chyle qualitative tests. The patient was discharged after 12-d persisting drainage, 3-d total parenteral nutrition and fasting, and other supportive treatments. No recurring symptoms were observed within 12 mo follow-up. CONCLUSION: Differential diagnosis is crucial for unusual thoracic effusion. Comprehensive diagnosis and treatment of chylothorax are necessary. Thorough intraoperative protection to relieve high thoracic pressure caused by the prone position is important.

A Minimally Invasive Exosome Spray Repairs Heart after Myocardial Infarction.

Myocardial infarction (MI) remains the most common cause of death worldwide. Many MI survivors will suffer from recurrent heart failure (HF), which has been recognized as a determinant of adverse prognosis. Despite the success of improved early survival after MI by primary percutaneous coronary intervention, HF after MI is becoming the major driver of late morbidity, mortality, and healthcare costs. The development of regenerative medicine has brought hope to MI treatment in the past decade. Mesenchymal stem cell (MSC)-derived exosomes have been established as an essential part of stem cell paracrine factors for heart regeneration. However, its regenerative power is hampered by low delivery efficiency to the heart. We designed, fabricated, and tested a minimally invasive exosome spray (EXOS) based on MSC exosomes and biomaterials. In a mouse model of acute myocardial infarction, EXOS improved cardiac function and reduced fibrosis, and promoted endogenous angiomyogenesis in the post-injury heart. We further tested the feasibility and safety of EXOS in a pig model. Our results indicate that EXOS is a promising strategy to deliver therapeutic exosomes for heart repair.

Acute cardiac complications and subclinical myocardial injuries associated with pheochromocytoma and paraganglioma.

BACKGROUND: Catecholamine excess arising from pheochromocytomas and paragangliomas (PPGLs) can cause a wide spectrum of cardiac manifestations, including acute cardiac complications (ACCs) and subclinical myocardial injuries (SMIs). In this study, we aimed to conduct a comprehensive analysis of ACCs and SMIs in a large cohort of patients with PPGLs. METHODS: We retrospectively analyzed the clinical data of consecutive patients with PPGLs admitted between January 2013 and July 2020 (n = 189). The prevalence of ACCs and SMIs and characteristics of patients identified with ACCs and SMIs were investigated. Moreover, comparisons were performed between patients with and without ACCs. RESULTS: Fourteen patients (7.4%) fulfilled the criteria for ACCs, including nine (4.8%) who presented with Takotsubo-like cardiomyopathy, four (2.1%) with heart failure with preserved ejection fraction, and finally one (0.5%) with catecholamine-induced cardiomyopathy. Compared to those without ACCs (n = 175), patients with ACCs had a higher prevalence of epinephrine-producing PPGLs (81.8% vs 33.9%, P = 0.006) and were more likely to show invasive behavior (61.5% vs 27.3%, P = 0.022) or hemorrhage/necrosis (53.9% vs 17.4%, P = 0.005) on histology. The apical sparing pattern (5/7, 71.4%) was the dominant impairment pattern of longitudinal strain (LS) for patients displaying Takotsubo-like cardiomyopathy. In patients without cardiac symptoms, a fairly high proportion (21/77, 27.3%) of patients who underwent screening for troponin and/or natriuretic peptide and/or echocardiography had SMIs. CONCLUSIONS: One in every fourteen PPGL patients presented with ACCs, and in the patients with Takotsubo-like cardiomyopathy, the apical sparing pattern was the primary impairment pattern of LS. Additionally, nearly one-third of patients without symptoms had SMIs. The diagnosis of PPGLs should be considered in patients with acute reversible cardiomyopathy, especially in those exhibiting an apical sparing pattern of LS.

Clinicopathological characteristics and prognostic factors for intrahepatic cholangiocarcinoma: a population-based study.

We aimed to explore the clinicopathological features and survival-related factors for intrahepatic cholangiocarcinoma (ICC). Eligible data were extracted from the Surveillance, Epidemiology and End Results (SEER) database from 2004 to 2015. Totally, 4595 ICC patients were collected with a male to female ratio of nearly 1:1. The higher proportion of ICC patients was elderly, tumor size ≥ 5 cm and advanced AJCC stage. Most patients (79.2%) have no surgery, while low proportion of patients receiving radiotherapy (15.1%). The median survival was 7.0 months (range 0-153 months). The 5-year CSS and OS rates were 8.96% and 7.90%. Multivariate analysis found that elderly age (aged ≥ 65 years old), male, diagnosis at 2008-2011, higher grade, tumor size ≥ 5 cm, and advanced AJCC stage were independent factors for poorer prognosis; while API/AI (American Indian/AK Native, Asian/Pacific Islander) race, married, chemotherapy, surgery and radiotherapy were independent favorable factors in both CSS and OS. Furthermore, stratified analysis found that chemotherapy and radiotherapy improved CSS and OS in patients without surgery. Age, sex, race, years of diagnosis, married status, grade, tumor size, AJCC stage, surgery, chemotherapy and radiotherapy were significantly related to prognosis of ICC. Chemotherapy and radiotherapy could significantly improve survival in patients without surgery.

Molecular Recognition and Imaging of Human Telomeric G-Quadruplex DNA in Live Cells: A Systematic Advancement of Thiazole Orange Scaffold To Enhance Binding Specificity and Inhibition of Gene Expression.

A series of fluorescent ligands, which were systematically constructed from thiazole orange scaffold, was investigated for their interactions with G-quadruplex structures and antitumor activity. Among the ligands, compound 3 was identified to exhibit excellent specificity toward telomere G4-DNA over other nucleic acids. The affinity of 3-Htg24 was almost 5 times higher than that of double-stranded DNA and promoter G4-DNA. Interaction studies showed that 3 may bind to both G-tetrad and the lateral loop near the 5'-end. The intracellular colocalization with BG4 and competition studies with BRACO19 reveal that 3 may interact with G4-structures. Moreover, 3 reduces the telomere length and downregulates hTERC and hTERT mRNA expression in HeLa cells. The cytotoxicity of 3 against cancer cells (IC50 = 12.7-16.2 µM) was found to be generally higher than noncancer cells (IC50 = 52.3 µM). The findings may support that the ligand is telomere G4-DNA specific and may provide meaningful insights for anticancer drug design.

A small-sized benzothiazole-indolium fluorescent probe: the study of interaction specificity targeting c-MYC promoter G-quadruplex structures and live cell imaging.

A small-sized c-MYC promoter G-quadruplex selective fluorescent BZT-Indolium binding ligand was demonstrated for the first time as a highly target-specific and photostable probe for in vitro staining and live cell imaging and it was found to be able to inhibit the amplification of the c-MYC G-rich sequence (G-quadruplex) and down-regulate oncogene c-MYC expression in human cancer cells (HeLa).

Genomic Analysis Reveals Novel Specific Metastatic Mutations in Chinese Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for more than 75% of renal cell carcinoma. Nearly 25% of ccRCC patients were diagnosed with metastasis. Though the genomic profile of ccRCC has been widely studied, the difference between localized and metastatic ccRCC was not clarified. Primary tumor samples and matched whole blood were collected from 106 sporadic patients diagnosed with renal clear cell carcinoma at Qilu Hospital of Shandong University from January 2017 to November 2019, and 17 of them were diagnosed with metastasis. A hybridization capture-based next-generation sequencing of 618 cancer-related genes was performed to investigate the somatic and germline variants, tumor mutation burden (TMB), and microsatellite instability (MSI). Five genes with significantly different prevalence were identified in the metastatic group, especially TOP1 (17.65% vs. 0%) and SNCAIP (17.65% vs. 0%). The altered frequency of PBRM1 (0% vs. 27%) and BAP1 (24% vs. 10%) differed between the metastatic and nonmetastatic groups, which may relate to the prognosis. Of these 106 patients, 42 patients (39.62%) had at least one alteration in DNA damage repair (DDR) genes, including 58.82% of metastatic ccRCC patients and 35.96% of ccRCC patients without metastasis. Ten pathogenic or likely pathogenic (P/LP) variants were identified in 11 sporadic clear cell renal cell carcinoma patients (10.38%), including rarely reported ATM (n=1), MUTYH (n=1), NBN (n=1), RAD51D (n=1), and BRCA2 (n=1). No significant difference in the ratio of P/LP variant carriers or TMB was identified between the metastatic and nonmetastatic groups. We found a unique genomic feature of Chinese metastatic ccRCC patients with a higher prevalence of alterations in DDR, TOP1, and SNCAIP. Further investigated studies and drug development are needed in the future.

Clinicopathological evidence of hepatitis B virus infection in the development of gastric adenocarcinoma.

Gastric cancer (GC) is one of the infection-related cancers. Helicobacter pylori and Epstein-Barr virus (EBV) were established risk factors for GC. Recently, there are several reports showing the inconsistent association between hepatitis B virus (HBV) infection and the development of GC. To explore the relationship between HBV infection and the development of GC, we designed a meta-analysis of previous epidemiological studies, a hospital-based case-control study, followed by an immunohistochemistry (IHC) assay of HBV-exposed GC samples. We found that HBV infection was associated with an increased risk of GC based on the meta-analysis. No significant association between HBV infection and GC was detected according to our hospital-based case-control study. Histological examination showed that the gastric epithelium positive for HBx demonstrated a higher nuclear-cytoplasmic ratio compared to those HBx-negative cells. HBx and HBcAg were expressed more in tumors than those in normal counterparts in HBV-exposed subjects, and PD-L1 was lower in GC tissues from HBV carriers than those in HBV clearances. In conclusion, HBV infection may contribute to a higher risk for GC based on the meta-analysis and to the morphological atypia of gastric epithelium by the histological assessment, and GC patients among HBV carriers showed lower expression of PD-L1 may lose the chance for immune checkpoint blockade therapy.

Signaling protein signature predicts clinical outcome of non-small-cell lung cancer.

BACKGROUND: Non-small-cell lung cancer (NSCLC) is characterized by abnormalities of numerous signaling proteins that play pivotal roles in cancer development and progression. Many of these proteins have been reported to be correlated with clinical outcomes of NSCLC. However, none of them could provide adequate accuracy of prognosis prediction in clinical application. METHODS: A total of 384 resected NSCLC specimens from two hospitals in Beijing (BJ) and Chongqing (CQ) were collected. Using immunohistochemistry (IHC) staining on stored formalin-fixed paraffin-embedded (FFPE) surgical samples, we examined the expression levels of 75 critical proteins on BJ samples. Random forest algorithm (RFA) and support vector machines (SVM) computation were applied to identify protein signatures on 2/3 randomly assigned BJ samples. The identified signatures were tested on the remaining BJ samples, and were further validated with CQ independent cohort. RESULTS: A 6-protein signature for adenocarcinoma (ADC) and a 5-protein signature for squamous cell carcinoma (SCC) were identified from training sets and tested in testing sets. In independent validation with CQ cohort, patients can also be divided into high- and low-risk groups with significantly different median overall survivals by Kaplan-Meier analysis, both in ADC (31 months vs. 87 months, HR 2.81; P <  0.001) and SCC patients (27 months vs. not reached, HR 9.97; P <  0.001). Cox regression analysis showed that both signatures are independent prognostic indicators and outperformed TNM staging (ADC: adjusted HR 3.07 vs. 2.43, SCC: adjusted HR 7.84 vs. 2.24). Particularly, we found that only the ADC patients in high-risk group significantly benefited from adjuvant chemotherapy (P = 0.018). CONCLUSIONS: Both ADC and SCC protein signatures could effectively stratify the prognosis of NSCLC patients, and may support patient selection for adjuvant chemotherapy.

RNF4 negatively regulates NF-κB signaling by down-regulating TAB2.

Most of NF-κB (nuclear factor kappa B) signaling molecules have various types of post-translational modifications. In this study, we focused on ubiquitination and designed a siRNA library including most ubiquitin-binding domains. With this library, we identified several candidate regulators of canonical NF-κB pathway, including RNF4. Overexpression of RNF4 impaired NF-κB activation in a dose-dependent manner, whereas RNF4 knockdown potentiated NF-κB activation. We showed that RNF4 interacts with the TAK1-TAB2-TAB3 complex, but not TAB1. Further, we found that RNF4 specifically down-regulated TAB2 through a lysosomal pathway, and knockdown of RNF4 impaired endogenous TAB2 degradation. Therefore, our findings will provide new insights into the negative regulation of NF-κB signaling.

Molecular engineering of Schiff-base linked covalent polymers with diverse topologies by gas-solid interface reaction.

The design and construction of molecular nanostructures with tunable topological structures are great challenges in molecular nanotechnology. Herein, we demonstrate the molecular engineering of Schiff-base bond connected molecular nanostructures. Building module construction has been adopted to modulate the symmetry of resulted one dimensional (1D) and two dimensional (2D) polymers. Specifically, we have designed and constructed 1D linear and zigzag polymers, 2D hexagonal and chessboard molecular nanostructures by varying the number of reactive sites and geometry and symmetry of precursors. It is demonstrated that high-quality conjugated polymers can be fabricated by using gas-solid interface reaction. The on-demanding synthesis of polymeric architectures with diverse topologies paves the way to fabricate molecular miniature devices with various desired functionalities.

Ubiquitin-associated domain-containing ubiquitin regulatory X (UBX) protein UBXN1 is a negative regulator of nuclear factor κB (NF-κB) signaling.

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.