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Pilomatricoma, a benign skin appendage tumor, also known as calcifying epithelioma, consists of islands of epithelial cells histologically that contain anucleated cells in the center surrounded by basophilic cells and partial calcification. Sporadic pilomatricomas commonly have somatic mutations in the gene CTNNB1, but causative genes from germline and the underlying pathophysiology are unclear. In this study, we identified a germline missense variant of PLCD1 encoding PLCδ1, c.1186G>A (p.Glu396Lys), in a large Chinese family with autosomal dominant multiple pilomatricomas. Phospholipase C, a key enzyme playing critical roles in intracellular signal transduction, is essential for epidermal barrier integrity. The p.Glu396Lys variant increased the enzymatic activity of PLCδ1, leading to protein kinase C/protein kinase D/extracellular signal–regulated kinase1/2 pathway activation and TPRV6 channel closure, which not only resulted in excessive proliferation of keratinocytes in vitro and in vivo but also induced local accumulation of calcium in the pilomatricoma-like tumor that developed spontaneously in the skin of Plcd1E396K/E396K mice. Our results implicate this p.Glu396Lys variant of PLCD1 from germline leading to gain-of-function of PLCδ1 as a causative genetic defect in familial multiple pilomatricomas.

Objective. This study is aimed at using genes related to the peroxisome proliferator-activated receptor (PPAR) pathway to establish a prognostic risk model in kidney renal clear cell carcinoma (KIRC). Methods. For this study, we first found the PPAR pathway-related genes on the gene set enrichment analysis (GSEA) website and found the KIRC mRNA expression data and clinical data through TCGA database. Subsequently, we used R language and multiple R language expansion packages to analyze the expression, hazard ratio analysis, and coexpression analysis of PPAR pathway-related genes in KIRC. Afterward, using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) website, we established the protein-protein interaction (PPI) network of genes related to the PPAR pathway. After that, we used LASSO regression curve analysis to establish a prognostic survival model in KIRC. Finally, based on the model, we conducted correlation analysis of the clinicopathological characteristics, univariate analysis, and multivariate analysis. Results. We found that most of the genes related to the PPAR pathway had different degrees of expression differences in KIRC. Among them, the high expression of 27 genes is related to low survival rate of KIRC patients, and the high expression of 13 other genes is related to their high survival rate. Most importantly, we used 13 of these genes successfully to establish a risk model that could accurately predict patients’ prognosis. There is a clear correlation between this model and metastasis, tumor, stage, grade, and fustat. Conclusions. To the best of our knowledge, this is the first study to analyze the entire PPAR pathway in KIRC in detail and successfully establish a risk model for patient prognosis. We believe that our research can provide valuable data for future researchers and clinicians.

Production of natural pertactin for pharmaceutical use is limited by its low abundance. In this study, recombinant pertactin was highly expressed in the form of inclusion bodies in E. coli . However, up to 75% of the soluble turned out as aggregates when refolding by pulse-fed batch dilution. The conceivable route for aggregate formation was proposed as that the C-terminus of partially folded intermediate with a strong hydrophobic core would intertwine with that region of newly added denatured protein, resulting in aggregation between proteins with different folding states. The key factor for prevention of aggregate formation was to improve the synchronization of refolding. For this purpose, flash-batch dilution was conducted at a scale of 5 L and achieved a monomeric refolding yield of above 70%. Aggregates formed were efficiently removed along with impurities by one-step chromatography of Ni-resin. The purity of monomeric pertactin was >98%. An overall yield was 320 mg per liter fermentation liquor with a total recovery of about 59%. The purified protein was characterized by MALDI-TOF, circular dichroism, fluorescence, HPLC and DSC, and showed similar physiochemical properties compared to its natural counterpart. Animal study showed similar immunological responses and antibodies elicited demonstrated a comparable reactivity.

Human ferritin (HFn) nanocaging is becoming an appealing platform for anticancer drugs delivery. However, protein aggregation always occurs during the encapsulation process, resulting in low production efficiency. A new approach using high hydrostatic pressure (HHP) was explored in this study to overcome the problem of loading doxorubicin (DOX) in HFn. At the pressure of 500MPa and pH 5.5, DOX molecules were found to be encapsulated into HFn. Meanwhile, combining it with an additive of 20mM arginine completely inhibited precipitation and aggregation, resulting in highly monodispersed nanoparticles with almost 100% protein recovery. Furthermore, stepwise decompression and incubation of the complex in atmospheric pressure at pH 7.4 for another period could further increase the DOX encapsulation ratio. The HFn-DOX nanoparticles (NPs) showed similar morphology and structural features to the hollow cage and no notable drug leakage occurred for HFn-DOX NPs when stored at 4°C and pH 7.4 for two weeks. HFn-DOX NPs prepared through HHP also showed significant cytotoxicity in vitro and higher antitumor bioactivity in vivo than naked DOX. Moreover, This HHP encapsulation strategy could economize on DOX that was greatly wasted during the conventional preparation process simply through a desalting column. These results indicated that HHP could offer a feasible approach with high efficiency for the production of HFn-DOX NPs.

Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5–4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity.

Increasing evidence has indicated that circular (circ)RNAs participate in carcinogenesis; however, the specific regulatory mechanisms underlying the effects of circRNAs, microRNAs (miRNAs/miRs) and genes on the development of clear cell renal cell carcinoma (CCRCC) remain unclear. In the present study, RNA microarray data from CCRCC tissues and control samples were downloaded from the Gene Expression Omnibus and The Cancer Genome Atlas, in order to identify significantly dysregulated circRNAs, miRNAs and genes. The Cancer-Specific circRNA Database was used to explore the interactions between miRNAs and circRNAs, whereas TargetScan and miRDB were employed to predict the mRNA targets of miRNAs. Functional enrichment and prognostic analyses were conducted in R. The results revealed that 324 circRNAs were downregulated, whereas 218 circRNAs were upregulated in cancer. In addition, a circRNA-miRNA-mRNA interaction network was constructed. Gene Ontology analysis of the upregulated genes revealed that these genes were enriched in biological processes, including ‘flavonoid metabolic process’, ‘cellular glucuronidation’ and ‘T cell activation’. The downregulated genes were mainly enriched in biological processes, such as ‘nephron development’, ‘kidney development’ and ‘renal system development’. The hub genes, including membrane palmitoylated protein 7, aldehyde dehydrogenase 6 family member A1, transcription factor AP-2α, collagen type IV α 4 chain, nuclear receptor subfamily 3 group C member 2, plasminogen, Holliday junction recognition protein, claudin 10, kinesin family member 18B and thyroid hormone receptor β, and the hub miRNAs, including miR-21-3p, miR-155-3p, miR-144-3p, miR-142-5p, miR-875-3p, miR-885-3p, miR-3941, miR-224-3p, miR-584-3p and miR-138-1-3p, were significantly associated with CCRCC survival. In conclusion, these results suggested that the significantly dysregulated circRNAs, miRNAs and genes identified in this study may be considered potential biomarkers of the carcinogenesis of CCRCC and the survival of patients with this disease.

MiR-27b downregulation is significantly associated with tamoxifen resistance in breast cancer cells. However, how it is downregulated in tamoxifen resistant (TamR) breast cancer cells and its downstream regulation were not clear. By performing MSP assay and QRT-PCR analysis with the use of 5-AZA-dC, a DNA methyltransferase inhibitor, we observed that TamR MCF-7 cells had significantly higher levels of methylation in the miR-27b promoter region than tamoxifen sensitive MCF-7 (TamS) cells and demethylation restored miR-27b expression. Re-expression of miR-27b sensitized TamR MCF-7 cells to tamoxifen, inhibited invasion and reversed epithelial-mesenchymal transition (EMT)-like properties. By using bioinformatics analysis and following dual luciferase and western blot analysis, this study confirmed a direct regulation of miR-27b on HMGB3 expression by binding to the 3′UTR. In addition, this study also found that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b in reducing tamoxifen resistance and cell invasion and in reversing EMT-like properties. Therefore, we infer that HMGB3 is a functional target of miR-27b in modulation of tamoxifen resistance and EMT.

// Yang Liu 1 , Ming Tian 1 , Hui Zhao 1 , Yue He 1 , Fengshuang Li 1 , Xiunan Li 1 , Xinping Yu 1 , Kuke Ding 3 , Pingkun Zhou 2 , Yumei Wu 1 1 Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100006, P.R.China 2 Department of Radiation Toxicology and Oncology, Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, P.R. China 3 National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, P.R. China Correspondence to: Yumei Wu, email: wym597118@163.com Keywords: IER5, cervical cancer, radiosensitivity, apoptosis, biomarker Received: December 20, 2016 Accepted: March 28, 2017 Published: April 05, 2017 ABSTRACT Purpose: To define the role of immediate-early 5 (IER5) gene as a promising biomarker in predicting the radiosensitivity and prognosis of cervical cancer patients receiving cisplatin-based concurrent chemoradiotherapy (DDP-CCRT). Results: Our investigations found that IER5 level was markedly elevated in cervical cancer patients after being treated with irradiation, which indicated IER5 was closely dose induced. By contrast, the correlation between IER5 and radiosensitivity cannot be confirmed by the present study. The up-regulation of IER5 expression effectively increased cell apoptosis after administration of irradiation ( P < 0.05). Using an ANOVA model for repeated-measures, we found significant association between the IER5 level and tumor size ( P < 0.05). Materials and Methods: Forty-three cervical cancer patients stage II b -III b received DDP-CCRT were registered. Biopsy tissues were obtained after administration of irradiation dose of 0 Gy, 2~6 Gy, 10 Gy, 20 Gy, 30 Gy, respectively. The IER5 protein and mRNA levels were measured by immunohistochemistry, western blot and quantitative polymerase chain reaction, respectively; besides, the apoptosis rate was assessed by transferase-mediated dUTP nick end labeling. Conclusions: Mechanistically, we confirmed that IER5 induced by radiation dose enhanced apoptosis of cervical cancer, was inversely associated with tumor size. In conclusion, our studies indicate target IER5 is improved to be a potential radiosensitizer for developing effective therapeutic strategies against cervical cancer to radiotherapy and a predictive biomarker for radiosensitivity.

Recent studies reported that long non-coding RNAs (lncRNAs) might play critical roles in regulating endocrine resistance of breast cancer. Urothelial carcinoma-associated 1 (UCA1) is an lncRNA with an oncogenic role in breast cancer. This study aimed to investigate whether UCA1 is involved in acquired tamoxifen resistance in estrogen receptor (ER)-positive cancer cells. Our findings reveal that tamoxifen induces UCA1 upregulation in ER-positive breast cancer cells in a HIF1α-dependent manner. UCA1 upregulation results in significantly enhanced tamoxifen resistance. The upregulated UCA1 sponges miR-18a, which is a negative regulator of HIF1α. Therefore, UCA1 upregulation is further enhanced through a miR-18a-HIF1α feedback loop. In addition, our data also showed that miR-18a is a modulator of tamoxifen sensitivity due to its regulative effect on cell cycle proteins. miR-18a inhibitor reduced the sensitivity of MCF-7 cells to tamoxifen, while miR-18a mimics sensitized BT474 cells to tamoxifen. Therefore, miR-18a downregulation also partly contributes to acquired tamoxifen resistance in the cancer cells. These findings provide some useful information for future clinical treatment of tamoxifen resistance.

Successfully recovering proinsulin's native conformation from inclusion body is the crucial step to guarantee high efficiency for insulin's manufacture. Here, two by-products of disulfide-linked oligomers and disulfide-isomerized monomers were clearly identified during proinsulin aspart's refolding through multiple analytic methods. Arginine and urea are both used to assist in proinsulin refolding, however the efficacy and possible mechanism was found to be different. The oligomers formed with urea were of larger size than with arginine. With the urea concentrations increasing from 2 M to 4 M, the content of oligomers decreased greatly, but simultaneously the refolding yield at the protein concentration of 0.5 mg/mL decreased from 40% to 30% due to the increase of disulfide-isomerized monomers. In contrast, with arginine concentrations increasing up to 1 M, the refolding yield gradually increased to 50% although the content for oligomers also decreased. Moreover, it was demonstrated that not redox pairs but only oxidant was necessary to facilitate the native disulfide bonds formation for the reduced denatured proinsulin. An oxidative agent of selenocystamine could increase the yield up to 80% in the presence of 0.5 M arginine. Further study demonstrated that refolding with 2 M urea instead of 0.5 M arginine could achieve similar yield as protein concentration is slightly reduced to 0.3 mg/mL. In this case, refolded proinsulin was directly purified through one-step of anionic exchange chromatography, with a recovery of 32% and purity up to 95%. All the results could be easily adopted in insulin's industrial manufacture for improving the production efficiency.