The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named “opvCRISPR” by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.

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BackgroundSplicing factor 3b subunit 4 (SF3B4) is a splicing factor and potential oncogene in hepatocellular carcinoma (HCC); however, its regulatory mechanism is yet unclear. We aimed to determine the role of SF3B4 in HCC and the underlying mechanism.MethodsTo investigate the association between alternative splicing events and miRNAs, putative miRNAs were screened using TargetScan. Expression levels of and prognostic information for SF3B4 and miRNAs were determined based on public genomic data and clinical samples. Then, we examined the possible roles of SF3B4 and miRNA-133b in HCC cells and a xenograft mouse model. Pearson correlation analysis and in vitro experiments verified SF3B4 as a miRNA-133b target. Protein levels of key targets from the SF3B4 signaling pathway were estimated using western blotting.FindingsThe expression of SF3B4 was upregulated in HCC tissues and cell lines whereas, the expression of miRNA-133b was downregulated. MiRNA-133b negatively regulated the expression of SF3B4. Effects of SF3B4 overexpression were partially abolished by miRNA-133b mimics, confirming that SF3B4 is a target of miRNA-133b. Moreover, molecules associated with SF3B4, including KLF4, KIP1, and SNAI2, were also modulated by miRNA-133b.InterpretationSF3B4 plays a crucial role in HCC and is negatively regulated by miRNA-133b. The miRNA-133b/ SF3B4 axis may serve as a new therapeutic target for HCC treatment.FundChina National Funds for Distinguished Young Scientists (No.81425019), the State Key Program of National Natural Science Foundation of China (No.81730076), Shanghai Science and Technology Committee Program (No.18XD1405300) and Specially-Appointed Professor Fund of Shanghai (GZ2015009). China National Funds for National Natural Science Fund (No.81672899).

Circulating tumor cells (CTCs) originate from tumor tissues and are associated with cancer prognosis. However, existing technologies for CTC detection are limited owing to a lack of specific or accurate biomarkers. Here, we developed a new method for CTC detection based on the karyoplasmic ratio, without biomarkers. Consecutive patients with liver cancer or non-cancer liver diseases were recruited. CTCs in blood samples were analyzed by imaging flow cytometry based on the karyoplasmic ratio as well as EpCAM and CD45. Microvascular invasion (MVI), tumor recurrence, and survival were recorded for all patients. A total of 56.2 ± 23.8/100,000 cells with high karyoplasmic ratios (HKR cells) were detected in cancer patients, which was higher than the number of HKR cells in the non-cancer group (7.6 ± 2.2/100,000). There was also a difference in HKR cells between liver cancer patients with and without MVI. Based on a receiver operating characteristic curve analysis, the threshold was 21.8 HKR cells per 100,000 peripheral blood mononuclear cells, and the area under the curve was higher than those of traditional methods (e.g., CD45 and EpCAM staining). These results indicate that the new CTC detection method was more sensitive and reliable than existing methods. Accordingly, it may improve clinical CTC detection.

Bone morphogenetic protein-9 (BMP9) shows great osteoinductive potential in bone regeneration. Periodontal ligament stem cells (PDLSCs) with multi-differentiation capability and low immunogenicity are increasingly used as seed cells for periodontal regenerative therapies. In the present study, we investigated the potent osteogenic activity of BMP9 on human PDLSCs (hPDLSCs), in which the c-Jun N-terminal kinase (JNK) pathway is possibly involved. Our results showed that JNK inhibition by the specific inhibitor SP600125 or adenovirus expressing small interfering RNA (siRNA) targeting JNK (AdR-si-JNK) significantly decreased BMP9-induced gene and protein expression of early and late osteogenic markers, such as runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN), in hPDLSCs. We also confirmed the in-vivo positive effect of JNKs on ectopic bone formation induced by hPDLSCs injected into the musculature of athymic nude mice and BMP9 ex vivo gene delivery. For the cellular mechanism, we found that BMP9 activated the phosphorylation of JNKs and Smad2/3, and that JNKs may engage in cross-talk with the Smad2/3 pathway in BMP9-mediated osteogenesis.

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The universal expression of various non-coding RNAs (ncRNAs) is now considered the main feature of organisms' genomes. Many regions in the genome are transcribed but not annotated to encode proteins, yet contain small open reading frames (smORFs). A widely accepted opinion is that a vast majority of ncRNAs are not further translated. However, increasing evidence underlines a series of intriguing translational events from the ncRNAs, which were previously considered to lack coding potential. Recent studies also suggest that products derived from such novel translational events display important regulatory functions in many fundamental biological and pathological processes. Here we give a critical review on the potential coding capacity of ncRNAs, in particular, about what is known and unknown in this emerging area. We also discuss the possible underlying coding mechanisms of these extraordinary ncRNAs and possible roles of peptides or proteins derived from the ncRNAs in disease development and theranostics. Our review offers an extensive resource for studying the biology of ncRNAs and sheds light into the use of ncRNAs and their corresponding peptides or proteins for disease diagnosis and therapy.

Objective: The dysregulation of ribosome biogenesis is associated with the progression of numerous tumors, including hepatocellular carcinoma (HCC). Small nucleolar RNAs (snoRNAs) regulate ribosome biogenesis by guiding the modification of ribosomal RNAs (rRNAs). However, the underlying mechanism of this process in HCC remains elusive. Methods: RNA immunoprecipitation and sequencing were used to analyze RNAs targeted by ribosome proteins. The biological functions of SNORA23 were examined in HCC cells and a xenograft mouse model. To elucidate the underlying mechanisms, the 2′-O-ribose methylation level of rRNAs was evaluated by qPCR, and the key proteins in the PI3K/Akt/mTOR pathway were detected using Western blot. Results: Twelve snoRNAs were found to co-exist in 4 cancer cell lines using RPS6 pull-down assays. SNORA23 was downregulated in HCC and correlated with the poor prognoses of HCC patients. SNORA23 inhibited the proliferation, migration, and invasion of HCC cells both in vitro and in vivo. We also found that SNORA23 regulated ribosome biogenesis by impairing 2′-O-ribose methylation of cytidine 4506 of 28S rRNA. Furthermore, SNORA23, which is regulated by the PI3K/Akt/mTOR signaling pathway, significantly inhibited the phosphorylation of 4E binding protein 1. SNORA23 and rapamycin blocked the PI3K/AKT/mTOR signaling pathway and impaired HCC growth in vivo. Conclusions: SNORA23 exhibited antitumor effects in HCC and together with rapamycin, provided a promising therapeutic strategy for HCC treatment. KEYWORDS SNORA23; ribosome biogenesis; rRNA methylation; HCC