A digital coding combination analysis for mutational genotyping using pyrosequencing.

In the present study, we developed a novel digital coding combination analysis (DCCA) to analyze the gene mutation based on the sample combination principle. The principle is that any numerically named sample is divided into two groups, any two samples are not grouped in the same two groups, and any sample can be tested within the detection limit. Therefore, we proposed a specific combination that N samples were divided into M groups. Then N samples were analyzed, which could obtain the mutation results of M mixed groups. If only two groups showed positive (mutant type) signals, the same sample number from two positive signal groups would be the positive sample, and the remaining samples were negative (wild type). If three groups or more exhibited positive results, the same sample number from three positive signal groups would be the positive sample. If some samples remained uncertain, individual samples could be analyzed on a small scale. In the present study, we used the two genotypes of a mutation site (A5301G) to verify whether it was a useful and promising method. The results showed that we could quantitatively detect mutations and demonstrate 100% consistent results against a panel of defined mixtures with the detection limit using pyrosequencing. This method was suitable, sensitive, and reproducible for screening and analyzing low-frequency mutation samples, which could reduce reagent consumption and cost by approximately 70-80% compared with conventional clinical methods.

A Novel Approach to the Bioluminescent Detection of the SARS-CoV-2 ORF1ab Gene by Coupling Isothermal RNA Reverse Transcription Amplification with a Digital PCR Approach.

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.

A Gene Expression Signature to Predict Nucleotide Excision Repair Defects and Novel Therapeutic Approaches.

Nucleotide excision repair (NER) resolves DNA adducts, such as those caused by ultraviolet light. Deficient NER (dNER) results in a higher mutation rate that can predispose to cancer development and premature ageing phenotypes. Here, we used isogenic dNER model cell lines to establish a gene expression signature that can accurately predict functional NER capacity in both cell lines and patient samples. Critically, none of the identified NER deficient cell lines harbored mutations in any NER genes, suggesting that the prevalence of NER defects may currently be underestimated. Identification of compounds that induce the dNER gene expression signature led to the discovery that NER can be functionally impaired by GSK3 inhibition, leading to synergy when combined with cisplatin treatment. Furthermore, we predicted and validated multiple novel drugs that are synthetically lethal with NER defects using the dNER gene signature as a drug discovery platform. Taken together, our work provides a dynamic predictor of NER function that may be applied for therapeutic stratification as well as development of novel biological insights in human tumors.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi.

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8 nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2 µM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5 µM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

IL-37 Is a Novel Proangiogenic Factor of Developmental and Pathological Angiogenesis.

OBJECTIVE: Angiogenesis is tightly controlled by growth factors and cytokines in pathophysiological settings. Interleukin 37 (IL-37) is a newly identified cytokine of the IL-1 family, some members of which are important in inflammation and angiogenesis. However, the function of IL-37 in angiogenesis remains unknown. We aimed to explore the regulatory role of IL-37 in pathological and physiological angiogenesis. APPROACH AND RESULTS: We found that IL-37 was expressed and secreted in endothelial cells and upregulated under hypoxic conditions. IL-37 enhanced endothelial cell proliferation, capillary formation, migration, and vessel sprouting from aortic rings with potency comparable with that of vascular endothelial growth factor. IL-37 activates survival signals including extracellular signal-regulated kinase 1/2 and AKT in endothelial cells. IL-37 promoted vessel growth in implanted Matrigel plug in vivo in a dose-dependent manner with potency comparable with that of basic fibroblast growth factor. In the mouse model of retinal vascular development, neonatal mice administrated with IL-37 displayed increased neovascularization. We demonstrated further that IL-37 promoted pathological angiogenesis in the mouse model of oxygen-induced retinopathy. CONCLUSIONS: Our findings suggest that IL-37 is a novel and potent proangiogenic cytokine with essential role in pathophy siological settings.

Molecular characterization of melanin-concentrating hormone (MCH) in Schizothorax prenanti: cloning, tissue distribution and role in food intake regulation.

Melanin-concentrating hormone (MCH) is a crucial neuropeptide involved in various biological functions in both mammals and fish. In this study, the full-length MCH cDNA was obtained from Schizothorax prenanti by rapid amplification of cDNA ends polymerase chain reaction. The full-length MCH cDNA contained 589 nucleotides including an open reading frame of 375 nucleotides encoding 256 amino acids. MCH mRNA was highly expressed in the brain by real-time quantitative PCR analysis. Within the brain, expression of MCH mRNA was preponderantly detected in the hypothalamus. In addition, the MCH mRNA expression in the S. prenanti hypothalamus of fed group was significantly decreased compared with the fasted group at 1 and 3 h post-feeding, respectively. Furthermore, the MCH gene expression presented significant increase in the hypothalamus of fasted group compared with the fed group during long-term fasting. After re-feeding, there was a dramatic decrease in MCH mRNA expression in the hypothalamus of S. prenanti. The results indicate that the expression of MCH is affected by feeding status. Taken together, our results suggest that MCH may be involved in food intake regulation in S. prenanti.

Current approaches to the diagnosis and treatment of white sponge nevus.

White sponge nevus (WSN) in the oral mucosa is a rare autosomal dominant genetic disease. The involved mucosa is white or greyish, thickened, folded and spongy. The genes associated with WSN include mutant cytokeratin keratin 4 (KRT4) and keratin 13 (KRT13). In recent years, new cases of WSN and associated mutations have been reported. Here, we summarise the recent progress in our understanding of WSN, including clinical reports, genetics, animal models, treatment, pathogenic mechanisms and future directions. Gene-based diagnosis and gene therapy for WSN may become available in the near future and could provide a reference and instruction for treating other KRT-associated diseases.

Appetite regulation in Schizothorax prenanti by three CART genes.

In recent years, cocaine- and amphetamine-regulated transcript (CART) has received much attention as mediators of appetite regulation in mammals. However, the involvement of CART in the feeding behavior of teleosts has not been well understood. In this study, three distinct CARTs were cloned from the Schizothorax prenanti (S. prenanti). Real-time quantitative PCR were applied to characterize the tissue distribution and appetite regulatory effects of CARTs in S. prenanti. The S. prenanti CART-1, CART-2 and CART-3 full-length cDNA sequences were 597 bp, 694 bp and 749 bp in length, encoding the peptides of 125, 120 and 104 amino acid residues, respectively. All the S. prenanti CARTs consisted of three exons and two introns. Tissue distribution analysis showed that the high mRNA levels of S. prenanti CART-1 were observed in the telencephalon and eye, followed by the hypothalamus, myelencephalon, and mesencephalon. The S. prenanti CART-2 mRNA was mainly found in the mesencephalon, hypothalamus, telencephalon and myelencephalon. The S. prenanti CART-3 mRNA was widely distributed among the tissues, with the high levels in the hypothalamus and foregut. In the periprandial experiment, all three CARTs mRNA expressions in the hypothalamus were highly elevated after a meal, suggesting that CARTs are postprandial satiety signals. In the fasting experiment, all three CARTs mRNA expressions decreased after fasting and increased after refeeding, suggesting that CARTs might be involved in regulation of appetite in the S. prenanti.

Leptin and cholecystokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression responses to periprandial changes and fasting.

In the present study, full-length cDNA sequences of leptin and cholecystokinin (CCK) were cloned from Schizothorax prenanti (S. prenanti), and applied real-time quantitative PCR to characterize the tissue distribution, and appetite regulatory effects of leptin and CCK in S. prenanti. The S. prenanti leptin and CCK full-length cDNA sequences were 1121 bp and 776 bp in length, encoding the peptide of 171 and 123 amino acid residues, respectively. Tissue distribution analysis showed that leptin mRNA was mainly expressed in the liver of S. prenanti. CCK was widely expressed, with the highest levels of expression in the hypothalamus, myelencephalon, telencephalon and foregut of S. prenanti. The CCK mRNA expression was highly elevated after feeding, whereas the leptin mRNA expression was not affected by single meal. These results suggested that CCK is a postprandial satiety signal in S. prenanti, but leptin might not be. In present study, leptin and CCK gene expression were both decreased after fasting and increased after refeeding, which suggested leptin and CCK might be involved in regulation of appetite in S. prenanti. This study provides an essential groundwork to further elucidate the appetite regulatory systems of leptin and CCK in S. prenanti as well as in other teleosts.

Schizothorax prenanti corticotropin-releasing hormone (CRH): molecular cloning, tissue expression, and the function of feeding regulation.

Corticotropin-releasing hormone (CRH) is a potent mediator of endocrine, autonomic, behavioral, and immune responses to stress. For a better understanding of the structure and function of the CRH gene and to study its effect on feeding regulation in cyprinid fish, the cDNA of the CRH gene from the brain of Schizothorax prenanti was cloned and sequenced. The full-length CRH cDNA consisted of 1,046 bp with an open reading frame of 489 bp encoding a protein of 162 amino acids. Real-time quantitative PCR analyses revealed that CRH was widely expressed in central and peripheral tissues. In particular, high expression level of CRH was detected in brain. Furthermore, CRH mRNA expression was examined in different brain regions, especially high in hypothalamus. In addition, there was no significant change in CRH mRNA expression in fed group compared with the fasted group in the S. prenanti hypothalamus during short-term fasting. However, CRH gene expression presented significant decrease in the hypothalamus in fasted group compared with the fed group (P < 0.05) on day 7; thereafter, re-feeding could lead to a significant increase in CRH mRNA expression in fasted group on day 9. The results suggest that the CRH may play a critical role in feeding regulation in S. prenanti.

The emerging roles of miRNA-mediated autophagy in ovarian cancer.

Ovarian cancer is one of the common tumors of the female reproductive organs. It has a high mortality rate, is highly heterogeneous, and early detection and primary prevention are very complex. Autophagy is a cellular process in which cytoplasmic substrates are targeted for degradation in lysosomes through membrane structures called autophagosomes. The periodic elimination of damaged, aged, and redundant cellular molecules or organelles through the sequential translation between amino acids and proteins by two biological processes, protein synthesis, and autophagic protein degradation, helps maintain cellular homeostasis. A growing number of studies have found that autophagy plays a key regulatory role in ovarian cancer. Interestingly, microRNAs regulate gene expression at the posttranscriptional level and thus can regulate the development and progression of ovarian cancer through the regulation of autophagy in ovarian cancer. Certain miRNAs have recently emerged as important regulators of autophagy-related gene expression in cancer cells. Moreover, miRNA analysis studies have now identified a sea of aberrantly expressed miRNAs in ovarian cancer tissues that can affect autophagy in ovarian cancer cells. In addition, miRNAs in plasma and stromal cells in tumor patients can affect the expression of autophagy-related genes and can be used as biomarkers of ovarian cancer progression. This review focuses on the potential significance of miRNA-regulated autophagy in the diagnosis and treatment of ovarian cancer.

Solvent-Responsive Magnetic Beads for Accurate Detection of SARS-CoV-2.

Although numerous approaches were proposed for the nucleic acid (NA)-based SARS-CoV-2 detection, the nonideal NA desorption efficiency of conventional magnetic beads (MBs) limits their widespread application. In this study, we developed solvent-responsive MBs (called responsive MBs), which, in the presence of buffers, modulated the absorption and desorption capacities of NA by flipping the surface -COO-. Relative to other commercial MBs, responsive MBs exhibited similar absorption profiles and markedly enhanced desorption profiles. When applied for NA detection of complex samples, responsive MBs exhibited better performance of RNA detection than DNA, with obvious advantages in sensitivity. Specifically, the RNA and DNA desorption rates of commercial MBs were ∼85 and 82.5%, while those of responsive MBs were nearly 94 and 93.5%, respectively. Furthermore, responsive MBs exhibited remarkable extraction ability in a wide range of tissues and better performance of RNA extraction than DNA. When applied for SARS-CoV-2 detection, the responsive MBs along with the simulated digital RT-LAMP (a previously established apparatus) further improved detection efficiency, yielding a precise quantitative detection as low as 25 copies and an ultimate sensibility detection of 5 copies/mL. It was also successfully employed in numerous NA-based technologies such as polymerase chain reaction (PCR), sequencing, and so on.

Development of a Novel Bioluminescence Pyrophosphate Assay for the High-Sensitivity Detection of Hepatitis B Virus.

The transmission of bloodborne viruses through transfusion remains a major blood supply-related safety concern, with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) being the most important pathogens in this context. Real-time bioluminescent pyrophosphate testing has been developed as a means of readily detecting bacterial cells within particular sample types without requiring the use of expensive or complex instrumentation. The sensitivity of this approach, however, is often limited such that it is not compatible with many potential applications. In this study, we sought to overcome the limitations of this pyrophosphate bioluminescent assay format by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) in place of dATP for PCR amplification, thereby dramatically reducing background signal levels. We leveraged this combination PCR and bioluminescent pyrophosphate assay approach to facilitate HBV detection. This assay yielded a limit of detection of 500 copies/mL, making it more sensitive than traditional bioluminescent assays, about 1000 times more sensitive than that of PCR product analysis by agarose gel electrophoresis, and roughly as sensitive as qPCR as a means of detecting viral DNA. We then used this assay to analyze 100 serum samples, with qPCR being used for result validation. The assay required 100 min to complete, and was able to detect as few as 500 copies/mL of viral DNA. Overall, our approach was rapid, sensitive, and simple, enabling users to readily detect HBV in a reliable and efficient manner.

Exploiting induced vulnerability to overcome PARPi resistance and clonal heterogeneity in BRCA mutant triple-negative inflammatory breast cancer.

Acquired resistance and clonal heterogeneity are critical challenges in cancer treatment, and the lack of effective computational tools hampers the discovery of new treatments to overcome resistance. Using high-throughput transcriptomic databases of compound perturbation profiles, we have developed a bioinformatic strategy for identifying candidate drugs to overcome resistance with combinatorial therapy. We devised this strategy during an investigation into the acquired resistance against PARP inhibitors (PARPi) in a triple-negative inflammatory breast cancer cell line. In this study, we derived multiple PARPi-resistant clones and characterized their transcriptomic adaptations compared to the parental clone. The transcriptomes of the resistant clones showed substantial heterogeneity, highlighting the importance of characterizing multiple clones from the same tumour. Surprisingly, we found that these transcriptomic changes may not actually confer PARPi resistance, but they may nevertheless induce a shared secondary vulnerability. By modeling our data in relation to transcriptomic perturbation profiles of compounds, we uncovered deficiencies in Ras signaling that resulted from transcriptional adaptation to long-term PARPi treatment across multiple resistant clones. Due to these induced deficiencies, we predicted that the resistant clones would be sensitive to pharmacological reinforcement of PARPi-induced transcriptional adaptation. We then experimentally validated this predicted vulnerability that is shared by multiple resistant clones. Our results thus provide a promising paradigm for integrating transcriptomic data with compound perturbation profiles in order to identify drugs that can exploit an induced vulnerability and overcome therapeutic resistance, thus providing another strategy towards precision oncology.

A novel bioluminescent approach to the loop-mediated isothermal amplification-based detection of Lactobacillus salivarius in feed samples.

Coupling loop-mediated isothermal amplification (LAMP) with a bioluminescent assay in real-time (LAMP-BART) is a strategy that can be readily leveraged to detect bacteria in particular samples of interest without the need for costly or complicated equipments. However, this approach exhibits poor sensitivity, and it additionally amplifies all target DNA including that derived from non-viable cells. Herein, we sought to overcome these traditional pyrophosphate bioluminescent assay limitations by utilizing 2-deoxyadenosine-5-(α-thio) -triphosphate (dATPαS) in place of dATP when conducting LAMP, thereby markedly reducing and stabilizing overall background signal levels, resulting in a detection limit of 3 CFU/µL. We were additionally able to ouple this LAMP-BART with propidium monoazide (PMAxx™) as a means of eliminating false-positive signals derived from nonviable cells. Herein, we detail the development of this PMAxx™-LAMP-BART assay and its use for the detection of live Lactobacillus salivarius. Our developed approach exhibited 100% specificity, with a 3 CFU/µL limit of detection (LOD) pure culture. In the application of feed, the LOD was 103 CFU per 10 g of spiked dry dog food and 102 CFU per 10 g of spiked chicken feed without enrichment. Traditional culture methods and a MALDI Biotyper were also used to confirm the accuracy of our novel assay system.

Effect of NELL1 on lung cancer stem­like cell differentiation.

The cancer stem cell theory recently has received enormous attention in cancer biology. Lung cancer stem­like cells are a subpopulation of undifferentiated lung tumor cells critical for lung cancer tumorigenesis, metastasis and resistance to therapy and disease relapse. The neural EGFL like 1 (NELL1) is a potent growth factor believed to preferentially target cells committed to the osteochondral lineage; yet, its expression and function in lung cancer are largely unknown. In the present study, we used specific medium to accumulate lung cancer stem­like cells of 95­D cells in spheres and obtained these highly expressed CD133 cells through flow cytometric cell sorting of CD133­stained cells which were termed 95­D lung cancer stem­like cells (95­D LCSCs). These 95­D LCSCs highly expressed stemness genes CD133, Oct4 and Sox2 determined by western blot analysis and quantitative real­time polymerase chain reaction (qPCR) analysis. Notably, we found that overexpression of NELL1 significantly reduced colony formation and invasion of 95­D LCSCs tested by soft agar colony formation and cell invasion assay. In addition, as determined by cell proliferation assay, overexpression of NELL1 increased the chemotherapeutic sensitivity of 95­D LCSCs to carboplatin and cisplatin. NELL1 also reduced the expression of phospho­MET (p­MET), Notch3 and HES1, which suggests that NELL1 may induce 95­D LCSC differentiation by inhibiting the expression of c­MET­Notch signaling. Our results suggest that NELL1 induces lung cancer stem­like cell differentiation, which provides a new potential therapeutic target for cancer stem cells.

Alternating morphology transitions in crystallization of NH4Cl on agar plates.

Two types of alternating morphology transitions have been observed in crystallization of NH4Cl on agar plates. One is the alternating morphology transitions between dense branching morphology and sparse branching morphology, and the other is the alternating morphology transitions between dense branching morphology and zigzag branching morphology. The appearance of them is found to depend on the mass proportion of agar to NH4Cl in the initial solution and the relative humidity. It is suggested that both the two alternating morphology transitions result from the oscillation of solute concentration in front of the growing interface caused by the competition of crystal growth and solute transfer at a moderate mass proportion. Which one of them occurs depends on the relative humidity, which controls the supersaturation.

Drug-induced premature senescence model in human dental follicle stem cells.

Aging is identified by a progressive decline of physiological integrity leading to age-related degenerative diseases, but its causes is unclear. Human dental pulp stem cells (hDPSCs) has a remarkable rejuvenated capacity that relies on its resident stem cells. However, because of the lack of proper senescence models, exploration of the underlying molecular mechanisms has been hindered. Here, we established a cellular model utilizing a hydroxyurea (HU) treatment protocol and effectively induced Human dental pulp stem cells to undergo cellular senescence. Age-related phenotypic changes were identified by augmented senescence-associated-ß-galactosidase (SA-ß-gal) staining, declined proliferation and differentiation capacity, elevated G0/G1 cell cycle arrest, increased apoptosis and reactive oxygen species levels. Furthermore, we tested the expression of key genes in various DNA repair pathways including nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. In addition, our results showed that Dental pulp stem cells from young donors are more resistant to apoptosis and exhibit increased non-homologous end joining activity compared to old donors. Further transcriptome analysis demonstrate that multiple pathways are involved in the HU-induced Dental pulp stem cells ageing, including genes associated with DNA damage and repair, mitochondrial dysfunction and increased reactive oxygen species levels. Taken together, the cellular model have important implications for understanding the molecular exploration of Dental pulp stem cells senescence and aging.

Interleukin 37 promotes angiogenesis through TGF-ß signaling.

IL-37 is a novel pro-angiogenic cytokine that potently promotes endothelial cell activation and pathological angiogenesis in our previous study, but the mechanisms behind the pro-angiogenic effect of IL-37 are less well understood. Extending our observations, we found that TGF-ß interacts with IL-37, and potently enhances the binding affinity of IL-37 to the ALK1 receptor complex, thus allowing IL-37 to signal through ALK1 to activate pro-angiogenic responses. We further show that TGF-ß and ALK1 are required in IL-37 induced pro-angiogenic response in ECs and in the mouse model of Matrigel plug and oxygen-induced retinopathy. The result suggests that IL-37 induces pro-angiogenic responses through TGF-ß, which may act as the bridging molecule that mediates IL-37 binding to the TGF-ß receptor complex.

Deubiquitinases in cancer.

Deubiquitinases are deubiquitinating enzymes (DUBs), which remove ubiquitin from proteins, thus regulating their proteasomal degradation, localization and activity. Here, we discuss DUBs as anti-cancer drug targets.