A strategy for precise and large scale identification of core fucosylated glycoproteins.

Core fucosylation (CF) patterns of some glycoproteins are more sensitive and specific than evaluation of their total respective protein levels for diagnosis of many diseases, such as cancers. Global profiling and quantitative characterization of CF glycoproteins may reveal potent biomarkers for clinical applications. However, current techniques are unable to reveal CF glycoproteins precisely on a large scale. Here we developed a robust strategy that integrates molecular weight cutoff, neutral loss-dependent MS(3), database-independent candidate spectrum filtering, and optimization to effectively identify CF glycoproteins. The rationale for spectrum treatment was innovatively based on computation of the mass distribution in spectra of CF glycopeptides. The efficacy of this strategy was demonstrated by implementation for plasma from healthy subjects and subjects with hepatocellular carcinoma. Over 100 CF glycoproteins and CF sites were identified, and over 10,000 mass spectra of CF glycopeptide were found. The scale of identification results indicates great progress for finding biomarkers with a particular and attractive prospect, and the candidate spectra will be a useful resource for the improvement of database searching methods for glycopeptides.

[Treatment of extensive cervicomandibular scar in children and adolescent patients with bilateral expanded scapular flaps: report of 7 consecutive cases].

PURPOSE: To explore the feasibility of applying bilateral free expanded scapular flaps to treat extensive cervicomandibular scar in children and adolescents. METHODS: This study reviewed 7 children and adolescent patients who received bilateral expanded scapular flaps to treat extensive cervicomandibular scars in the Pediatric Plastic Surgery Ward from August 2018 to December 2020. The scars in all patients involved neck, mandible, and anterior chest. The cervical scars involved the anterior neck and one or both sides of the lateral neck, and there were varying degrees of cervical dysfunction and mandibular dysplasia. The operation was completed into two stages. In the first stage, the expanded circumflex scapular artery perforator flaps were designed on both sides of the back and soft tissue expanders were implanted. The expansion process lasted for 6-14 months. In the second stage, the scar tissue was removed and contracture was released, and the expanded flaps were harvested. The cervical wound was repaired with free flap transplantation by anastomosing the facial artery and vein with the circumflex scapular artery and vein. The donor sites were closed directly. RESULTS: In this series of 7 patients, one patient had poorly healed incision after the expander was implanted. One expanded flap ruptured before the second-stage surgery, which was successfully treated by secondary surgery. One patient had expansion problem due to the blockage of the internally placed injection bottle, which was treated by placing the injection bottle externally. One patient developed a small area of ischemic necrosis at the distal end of the flap after transplantation, which was treated conservatively with dressing change. The postoperative follow-up was 6 months to 2 years. The cervico-mandibular angle restored to normal range, the cervical extension, flexion, and rotation were significantly improved. Two patients underwent flap thinning and scar releasing. CONCLUSIONS: The route of the circumflex scapular artery is constant. Bilateral expanded scapular flap transplantation can be used to repair extensive cervicomandibular scar in children and adolescent patients. The flap donor site is concealed and secondary damage is minimal.

Circ_ANKIB1 stabilizes the regulation of miR-19b on SOCS3/STAT3 pathway to promote osteosarcoma cell growth and invasion.

Osteosarcoma is a highly malignant tumor. The molecular mechanism of its occurrence and development has not yet been clarified. Current studies have found that noncoding RNAs, such as circular RNAs (circRNAs) and microRNAs (miRNAs), play important regulatory roles in the progression of diseases. Our previous studies have shown that miR-19b is an oncogene in osteosarcoma. Further studies have shown that circ_ANKIB1 has binding sites for miR-19b, and both molecules were generally upregulated in osteosarcoma cells. RIP assay, RNA pull down, and dual-luciferase reporter gene assay showed that circ_ANKIB1 could directly bind to miR-19b and act as an miR-19b sponge in osteosarcoma cells. Circ_ANKIB1 promoted miR-19b expression, inhibited the expression of the downstream target gene SOCS3, and then activated the STAT3 pathway. When cotransfected with circ_ANKIB1 siRNA, and miR-19b mimics, the expression of SOCS3 and the phosphorylation level of STAT3 did not change significantly. Continuous detection of cell growth and invasion showed that the downregulation of circ_ANKIB1 or miR-19b significantly inhibited cell proliferation and invasion, but increased the apoptotic level. When cotransfected with circ_ANKIB1 siRNA and miR-19b mimics or SOCS3 siRNA, the cell proliferation, apoptosis, and invasion levels did not change significantly, suggesting that circ_ANKIB1 could affect the STAT3 pathway and osteosarcoma cell growth and invasion by enhancing the regulation of miR-19b on the downstream target gene SOCS3. These findings suggest that circRNAs stabilize miRNA functions, and further studies on this new function of circRNAs will provide a meaningful reference for the diagnosis and treatment of tumors and other diseases.

Puerarin alleviates streptozotocin (STZ)-induced osteoporosis in rats through suppressing inflammation and apoptosis via HDAC1/HDAC3 signaling.

Diabetic osteoporosis is a severe public health concern in the world. Puerarin (PU) is extensively attractive due to its superior bioactivities. In the study, we found that PU protected against streptozotocin (STZ)-induced osteoporotic changes in rats. PU treatment improved STZ-induced diabetes in rats, as evidenced by the reduced serum glucose and insulin levels. PU administration markedly attenuated bone loss and tartarate-resistant acid phosphatase (TRAP) activity in STZ-induced rats. Bone mineral density (BMD) was significantly decreased in diabetic rats, while being prevented by PU. STZ-induced impairments in microarchitecture of femoral tissues were markedly alleviated by PU treatment. In addition, bone-specific alkaline phosphatase (BALP), osteoprotegerin (OPG) and Runt-related transcription factor 2 (Runx2) levels in serum or tibia were improved by PU in STZ-injected rats; however, TRACP isoform 5b (TRACP-5b), carboxy-terminal collagen cross-links (ß-CTX) and receptor activator of nuclear factor-κB ligand (RANKL) levels were decreased. Further, PU treatment inhibited inflammation and apoptosis in STZ-treated rats. Additionally, STZ injection increased histone deacetylase (HDAC)-1 and -3 expressions in femoral heads of rats, which were relieved by PU treatment. Notably, both HDAC1 and HDAC3 could enhance osteoporosis in vitro, as proved by the decreased ALP and Runx2 levels and the increased TRAP expression. Inflammation and apoptosis were exacerbated by HDAC1/3 over-expression, which were markedly diminished by PU treatment. In contrast, suppressing HDAC1/3 significantly abrogated fructose (Fru)-elicited inflammation and apoptosis in cells. Collectively, our data illustrated that PU is a potential therapeutic option to prevent diabetic osteoporosis by inhibiting HDAC1/HDAC3 signaling.

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

Novel nucleolar isolation method reveals rapid response of human nucleolar proteomes to serum stimulation.

The nucleolus is the location of ribosomal biogenesis, and plays crucial regulatory roles in nuclear responses to stress. Here, we report a new and improved nucleolar isolation method, which is simpler and more efficient than the traditional method. The purity of nucleoli obtained by using the new protocol is comparable to that by using the classical method, as judged by electron microscopy, Western blotting and SILAC-based quantitative proteomics. Moreover, the improved efficiency of cell harvesting in the new method, biochemical events in the nucleolus could be "frozen" and captured at precisely controlled time points. Time-lapse nucleolar proteomics after serum stimulation in HeLa cell revealed for the first time that some nucleolar proteins respond to serum stimulation within a time period as short as the first 5 min of serum re-stimulation. Proteins involved in ribosomal biogenesis and in DNA damage repair are among the most dynamic proteins during the first 10 min after serum replenishment. Notably, the proliferation marker Ki-67 is also found to enter the nucleolus after serum replenishment. To our knowledge, this is the first study that demonstrates such fast responses in the nucleolus, further confirming the rapid plasticity of this organelle.