Colorimetry-Based Phosphate Measurement for Polymerase Elongation.

DNA detection, which includes the measurement of variants in sequences or the presence of certain genes, is widely used in research and clinical diagnosis. Both require DNA-dependent DNA polymerase-catalyzed strand extension. Currently, these techniques rely heavily on the instruments used to visualize the results. This study introduced a simple and direct colorimetric method to measure polymerase-directed elongation. First, pyrophosphate (PPi), a by-product of strand extension, is converted into phosphate (Pi). Phosphate levels were measured using either Mo-Sb or BIOMOL Green reagent. This study showed that this colorimetry can distinguish single-base variants and detect PCR products in preset stringent conditions, implicating the potential value of this strategy to detect DNA.

New role of gramicidin A in RIG-I-like receptors-mediated IFN signalling.

The pattern recognition receptors (PRRs) sense exogenous molecular patterns most commonly derived from invading pathogens, to active the interferon (IFN) signalling. In the cytoplasm, the viral double-stranded RNAs (dsRNAs) are sensed by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), depending on the length and chemical properties. Through the binding and oligomerizing onto the RNAs, they form filament to initiate the signalling cascade. Regulation of these receptors' activities are essential for manipulating the strength of IFN signalling. Here, through the virtual screening of chemical reagents using the published MDA5-dsRNA complex structure (PDB: 4GL2), we identified an antibiotic, gramicidin A as a stimulator that enhanced MDA5-mediated IFN signalling. Cytotoxic assay and IFN signalling assay suggested that disruption of lipid membrane, which is a well-defined mechanism of gramicidin A to perform its action, was dispensable in this process. Sucrose gradient ultracentrifugation assay showed that the gramicidin A treatment enhanced MDA5 oligomerization status in the presence of dsRNA. Our work implicated a new role of gramicidin A in innate immunity and presented a new tool to manipulate MDA5 activity.

Type I Interferon-Induced TMEM106A Blocks Attachment of EV-A71 Virus by Interacting With the Membrane Protein SCARB2.

Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the main causative agents of hand, foot and mouth disease (HFMD) worldwide. Studies showed that EV-A71 and CV-A16 antagonize the interferon (IFN) signaling pathway; however, how IFN controls this viral infection is largely unknown. Here, we identified an IFN-stimulated gene, Transmembrane Protein 106A (TMEM106A), encoding a protein that blocks EV-A71 and CV-A16 infection. Combined approaches measuring viral infection, gene expression, and protein interactions uncovered that TMEM106A is required for optimal IFN-mediated viral inhibition and interferes with EV-A71 binding to host cells on the receptor scavenger receptor class B member 2 (SCARB2). Our findings reveal a new mechanism contributing to the IFN-mediated defense against EV-A71 and CV-A16 infection and provide a potential strategy for HFMD treatment by using the antiviral role of TMEM106A against enterovirus.

Emerging Roles of lncRNAs Regulating RNA-Mediated Type-I Interferon Signaling Pathway.

The type-I interferon (IFN-I) signaling pathway plays pivot roles in defending against pathogen invasion. Exogenous ssRNA and dsRNA could be immunogenic. RNA-mediated IFN signaling is extensively studied in the field. The incorrect functioning of this pathway leads to either autoimmune diseases or suffering from microorganism invasion. From the discrimination of "self" and "non-self" molecules by receptors to the fine-tune modulations in downstream cascades, all steps are under the surveillance featured by complex feedbacks and regulators. Studies in recent years highlighted the emerging roles of long noncoding RNAs (lncRNAs) as a reservoir for signaling regulation. LncRNAs bind to targets through the structure and sequence, and thus the mechanisms of action can be complex and specific. Here, we summarized lncRNAs modulating the RNA-activated IFN-I signaling pathway according to the event order during the signaling. We hope this review help understand how lncRNAs are participating in the regulation of IFN-I signaling.

Long Noncoding RNA XIST Acts as a ceRNA of miR-362-5p to Suppress Breast Cancer Progression.

Background: Long noncoding RNAs (lncRNAs) X inactivate-specific transcripts (XIST) have been found to be dysregulated in breast cancer (BC). Nevertheless, the influence and mechanism of XIST on BC progression remain largely undefined. Methods: The expression levels of XIST, miR-362-5p, and ubiquitin-associated protein 1 (UBAP1) mRNA were detected by quantitative real-time polymerase chain reaction. Cell proliferation, apoptosis, migration, and invasion abilities were determined using MTT assay, flow cytometry, and transwell assay, respectively. Targeted relationship between miR-362-5p and XIST or UBAP1 was validated by the dual-luciferase reporter assay. Western blot was performed to evaluate UBAP1 protein level. Xenograft mice model was established for the investigation of XIST in tumor growth. Results: The authors' data indicated that XIST and UBAP1 were downregulated in BC tissues and cells. XIST overexpression weakened BC cell proliferation, migration, invasion, and facilitated the apoptosis, and XIST silencing exerted opposite effect. Mechanistically, XIST directly interacted with miR-362-5p and miR-362-5p mediated the regulatory effects of XIST overexpression on BC cell malignant behaviors. UBAP1 was a direct target of miR-362-5p. MiR-362-5p exerted its regulatory effects on BC cell behaviors by UBAP1. Moreover, XIST modulated UBAP1 expression through acting a competing endogenous RNA of miR-362-5p. XIST overexpression mediated antiproliferation, antimigration, anti-invasion, and proapoptosis effects were abated by the restored expression of UBAP1 in BC cells. Furthermore, the upregulation of XIST hindered tumor growth in vivo. Conclusion: The current study suggested that XIST overexpression hampered BC cell progression in vitro and in vivo at least partially by targeting the miR-362-5p/UBAP1 axis, illuminating XIST as a promising therapeutic agent for BC management.

Cooperatively transcriptional and epigenetic regulation of sonic hedgehog overexpression drives malignant potential of breast cancer.

Sonic hedgehog (Shh), a ligand of Hedgehog signaling pathway, is considered an important oncogene and an exciting potential therapeutic target in several cancers. Comprehensive understanding of the regulation mechanism of Shh in cancer cells is necessary to find an effective approach to selectively block its tumorigenic function. We and others previously demonstrated that nuclear factor-kappa B (NF-κB) activation and promoter hypomethylation contributed to the overexpression of Shh. However, the relationship between transcriptional and epigenetic regulation of Shh, and their roles in the malignant phenotype of cancer cells are still not clearly elucidated. In the present study, our data showed that the level of Shh was higher in breast cancer tissues with positive NF-κB nuclear staining and promoter hypomethylation. In addition, survival analysis revealed that Shh overexpression, but not hypomethylation and NF-κB nuclear staining, was a poor prognosis indicator for breast cancers. Moreover, in vitro data demonstrated that both NF-κB activation and hypomethylation in promoter region were positively associated with the overexpression of Shh. Mechanistically, the hypomethylation in Shh promoter could facilitate NF-κB binding to its site, and subsequently cooperate to induce transcription of Shh. Furthermore, the biological function data indicated that overexpressed Shh enhanced the self-renewal capacity and migration ability of breast cancer cells, which could be augmented by promoter demethylation and NF-κB activation. Overall, our findings reveal multiple and cooperative mechanisms of Shh upregulation in cancer cells, and the roles of Shh in tumor malignant behavior, thus suggesting a new strategy for therapeutic interventions to reduce Shh in tumors and improve patients' prognosis.

[Operative experiences on transumbilical single incision laparoscopic cholecystectomy].

OBJECTIVE: To study the feasibility and safety of transumbilical single incision laparoscopic cholecystectomy. METHODS: Eighty-two cases with gallbladder diseases were underwent with transumbilical single incision laparoscopic cholecystectomy. Some difficulties and countermeasure in operations were analyzed. RESULTS: Nine in all patients were converted because of the surrounding inflammation of gallbladder, difficult to dissect in Calot's triangulation and variation of gallbladder artery. Of them, 6 cases converted to two-hole laparoscopic cholecystectomy and 3 cases to open operation. Other 73 cases were successfully operated with transumbilical single incision laparoscopic cholecystectomy. The success rate was 89.0%. The average operative duration was 48.4 min. The average operative blood loss was 20.8 ml. Patients returned to liquid food on the first day after operation. Hospital duration was 3 ~ 6 d. During 13.7 months of follow-up, there was no bile duct injury, large bleeding, incision infection, bile fistula and umbilical hernia. The incision healed well. The scar in umbilicus was concealing and difficult to be observed. CONCLUSIONS: Transumbilical single incision laparoscopic cholecystectomy is safe and feasible. The difficulties in technique are easy to be broken through.

[Clinical study of laparoscopic rectal surgery without abdominal scar].

OBJECTIVE: To assess the feasibility and safety of laparoscopic rectal surgery without abdominal scar. METHODS: A total of 23 patients of rectal cancer were operated by laparoscopy with the assistance of PPH (procedure for prolapse and hemorrhoids) anal expander and TEM (transanal endoscopic microsurgery) outer-shell according to the principle of TME (total mesorectal excision). RESULTS: All operations were successfully accomplished laparoscopically. There was no conversion into an open procedure. The average operative duration was 129 (105 - 211) minutes. The intra-operative blood loss volume was 152 (85 - 420) ml. No immediate or delayed injury of urinary duct and other intra-operative severe complications, such as massive hemorrhage of presacral venous plexus, occurred. There was no pelvic infection or anastomotic stoma fistula during the post-operative period. The average follow-up period was 13.4 months. Neither anastomotic stoma recurrence nor Trocar implantation occurred. CONCLUSION: The laparoscopic technique of hybrid NOTES (Natural Orifice Transluminal Endoscopic Surgery), plus PPH/TEM is both feasible and convenient for selective rectal cancer surgery. There is no need for extra abdominal incision. It is recommended for laparoscopic rectal surgery.

[The anatomical characteristics and its applied significance of laparoscopic rectal surgery].

OBJECTIVE: To realize the anatomical characteristics of laparoscopic rectum surgery and its clinical significance. METHODS: 117 cases with benign and malignant diseases of rectum were operated by laparoscopic methods. The anatomy closely related with operation was analyzed. RESULTS: The median operative time were 144 min. 4 cases were converted to open operations, so the converted rate was 1.7%. The blood loss in operation was rather little with an average of 126 ml. No instant or delayed injury of ureters, large bleeding in front of sacrum and other operation-related severe complications happened intra- and after operation. Dissecting only in one case disrupted the anterior-left wall of rectum. CONCLUSION: Laparoscopic rectal surgery is clinically feasible. Mastering the anatomical characteristics of laparoscopic rectum surgery can make us reduce the operative mistakes and complications.

[Standardized skill of mastoscopic axillary lymph node dissection].

OBJECTIVE: To investigate the standardized skill of mastoscopic axillary lymph node dissection (MALND). METHODS: The clinical results and operation experiences of 473 mastoscopic lymph node dissections performed were analyzed. RESULT: The operation time was 22 - 156 minutes with the average of 42. The operative bleeding was minimal. No case was converted to conventionally open operation because of the uncontrolled bleeding. Fourteen (4 - 38) lymph nodes were removed. There was no intra- or post-operative complication. CONCLUSION: The special view of mastoscopic lymph node dissection makes operative dissection clear. The suitable procedure and standardization of the operative method can stride over the learning curve, so as to quicken the operation and avoid the operative complication.