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The research on the morphological and mechanical properties of single cells has provided a crucial way of understanding the cellular physiology and metabolism. In this study, the selective anticancer effects of Phellinus linteus on A431 and HaCat cells and their morphological and mechanical properties were systematically investigated by atomic force microscopy (AFM). Notably, the cell morphology on the micronano scale was observed under both the physiological environment and immobilization conditions. The significant morphological changes of A431 cells from the flat to spherical shape, the increase of cell height, and the decrease of the particles on the cell membrane were confirmed to be related to the cell apoptosis under the treatment of the Phellinus linteus water extract (PLWE). Moreover, the small morphology variations of HaCat cells showed that the PLWE presented a high anticancer effect on A431 cells but low toxicity on HaCat cells, which indicated a potential cell selectivity between cancer and normal cells. This work proved that Phellinus linteus could be used as a potential candidate for selective anticancer treatments.

Drug resistance is an increasing problem in the treatment of vaginitis bacterial vaginosis and vulvovaginal candidiasis caused by biofilm-forming bacteria and fungi. In this study, the antimicrobial activities of the synergistic effects coordinated by antimicrobial peptides HPRP-A1, HPRP-A2, and chlorhexidine acetate (CHA), against biofilms caused by Gram-negative and Gram-positive bacteria, and one fungus Candida albicans, were investigated in vitro and in mouse and rat infection models. The minimal biofilm inhibitory concentration and the fractional biofilm inhibitory concentration of HPRP-A1, HPRP-A2 alone and in combination with CHA were determined by classical methods. The crystal violet staining assay was used to determine the biomass of agents on biofilms. The MTT assay was used to determine the metabolic activity of agents on biofilms. Different microscopy assays were used to observe the morphological characteristics of bacterial and fungal biofilms after drug treatments. The extracellular polysaccharide was detected by a phenol–sulfuric acid method. The combined uses of HPRP-A1, HPRP-A2 and CHA exerted strong synergistic effects to inhibit the formation of bacterial and fungal biofilms and disrupt the construction of biofilms in vitro and in vivo. In animal vaginitis models, the drug combination reduced 99.9% of the number of bacterial and fungal CFU, and showed significant therapeutic effects against biofilm vaginitis. The mechanism of membrane disruption coordinated by the combination of drugs depends not only on the inhibition of extracellular polysaccharides secreted by bacterial and fungal cells, but also on the ability of the peptides to penetrate the cell membrane.

Conjugation of cell-penetrating peptides to anticancer peptides is an effective strategy to enhance tumor treatment. The hybrid peptide, kla-TAT, by attaching the TAT peptide to the C-terminus of the pro-apoptotic peptide kla exhibited strong anticancer activity when co-administration with other peptide, but the systematic mechanism was not clear. In this study, the mechanisms of action of kla-TAT including the uptake pathway, distribution in cytoplasm, apoptosis-inducing ability and micro-morphology were investigated. The results indicated that the hybrid peptide internalized into A549 cells through two transmembrane mechanisms: endocytosis mediated by the clathrin-mediated endocytosis route and the rapid membrane disruption mechanism. Peptides that were endocytosed could interact with the mitochondrial membrane, while peptides internalized by disrupting the cell membrane caused disruption of the mitochondrial membrane. Thus, the hybrid kla-TAT peptide transfers through the cell membrane, inducing apoptosis by destroying the mitochondrial membrane and causethe expression of cyclin-D1 down-regulation. Meanwhile, the changes of the micro-morphology of the cancer cells was detected using atomic force microscope (AFM) and scanning electron microscope (SEM), which provided the visualized evidence for the mechanism of the peptides.

Jie Zhu,1,2 Yibing Huang,1,2 Mingxia Chen,3 Cuihua Hu,4 Yuxin Chen1–3 1Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, People’s Republic of China; 2College of Life Sciences, Jilin University, Changchun, People’s Republic of China; 3Jiangsu ProteLight Pharmaceutical & Biotechnology Co., Ltd., Jiangyin, People’s Republic of China; 4International Research Centre for Nano Handling and Manufacturing, Changchun University of Science and Technology, Changchun, People’s Republic of ChinaCorrespondence: Yuxin ChenKey Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, No. 2699, Qianjin St., Changchun 130021, People’s Republic of ChinaTel/fax +86 431 8515 5200Email chen_yuxin@jlu.edu.cnBackground and purpose: To reduce the resistance and allergic reaction to chlorhexidine acetate (CHA) in the current treatment of (Bacterial vaginosis) BV and (vulvovaginal candidiasis) VVC in female vaginitis. In this study, the antimicrobial activities and mechanism of action of the synergistic effects of antimicrobial peptides (AMPs) HPRP-A1 and HPRP-A2, and CHA, against Gram-negative and Gram-positive bacteria, and one fungus Candida albicans (C. albicans) were investigated in vitro and in mouse and rat vaginitis infection models in vivo.Results: HPRP-A1, HPRP-A2 and CHA showed significant synergistic effects on the antimicrobial activities against different Gram-negative and Gram-positive bacteria and C. albicans. The combined application of HPRP-A2 and CHA exhibited strong synergistic effects in the mouse and rat vaginitis models caused by bacteria or C. albicans.Conclusion: This study may prompt the development of new drug combinations against vaginitis infections, including mixed bacterial and fungal infections and multi-drug-resistant infections.Keywords: antimicrobial peptide, chlorhexidine acetate, antibacterial, antifungal, vaginitis

Conductive atomic force indentation (CAFI) was proposed to study the self‐repair behaviour of the neuronal cell membrane here. CAFI was used to detect the changes of membrane potentials by performing the mechanical indentation on neurons with a conductive atomic force microscope. In the experiment, a special insulation treatment was made on the conductive probe, which turned out to be a conductive nanoelectrode, to implement the CAFI function. The mechanical properties of the neuronal cell membrane surface were tested and the membrane potential changes of neurons cultured in vitro were detected. The self‐repair behaviour of the neuronal cell membrane after being punctured was investigated. The experiment results show that CAFI provides a new way for the study of self‐repair behaviours of neuronal cell membranes and mechanical and electrical properties of living cells.

In this work, the conductivity of DNA molecules under the effect of magnetic fields was studied by conductive atomic force microscopy (C-AFM). It is found that the self-assembly images of $7.5\ \text{ng}/\mu \mathrm{L}$ DNA aqueous solution on the bare mica and Au layer surfaces are quite different, and the DNA molecules are obviously stretched on the Au layer surface and single stretched DNA strands were obtained. In addition, the conductivity of single stretched DNA molecules in the horizontal stable magnetic field (SMF) and high frequency electromagnetic field (HFEF) were investigated in detail. The results showed that the conductivity of DNA was weakened by the effect of magnetic field.

In this work, a rich variety of self-assembled DNA patterns were obtained in the magnetic field. Herein, atomic force microscopy (AFM) was utilized to investigate the effects of the concentration of DNA solution, intensity and direction of magnetic field and modification of mica surface by different cations on the self-assembly of DNA molecules. It was found that owning to the change of the DNA concentration, even under the same magnetic field, the DNA self-assembly results were different. The in situ test results showed that the DNA self-assembly in an magnetic field was more likely to occur in liquid phase than in gas phase. In addition, whether in a horizontal or vertical magnetic field, a single stretched dsDNA was obtained in a certain DNA concentration and magnetic field intensity. Besides, the modification of cations on the mica surface significantly increased the force between the DNA molecules and mica surface, and further changed the self-assembly of DNA molecules under the action of magnetic field.

Conductive atomic force microscopy (C-AFM) is a powerful tool used in the microelectronics analysis by applying a certain bias voltage between the conducting probe and the sample and obtaining the electrical information of sample. In this work, the surface morphological information and current images of the lambda DNA (

Atomic force microscopy (AFM) can be used to obtain the physical information of single live cancer cells; however, the physical changes in live cells with time based on AFM remain to be studied, which play a key role in the evaluation of the efficacy and side effects of drugs. Herein, the treatment of the A549 cell line with the anticarcinogen 5-fluorouracil has been discussed based on the AFM analysis of their continuous physical changes, including their surface morphology, height, adhesion and Young's modulus, with time. In comparison, the African green monkey kidney (Vero) cell line was tested as normal cells to determine the side effects of 5-fluorouracil. The results show that the optimal concentration of 5-fluorouracil is about 500 μM, which presents the best anticancer effect and mild side effects.

The chimeric peptide HPRP-A1-iRGD, composed of a chemically conjugated tumor-homing/penetration domain (iRGD) and a cationic anticancer peptide domain (HPRP-A1), was used to study the effect of targeted modification to enhance the peptide's specificity, penetration, and tumor accumulation ability. The iRGD domain exhibits tumor-targeting and tumor-penetrating activities by specifically binding to the neuropilin-1 receptor. Acting as a homing/penetration domain, iRGD contributed to enhancing the tumor selectivity, permeability, and targeting of HPRP-A1 by targeted receptor dependence. As the anticancer active domain, HPRP-A1 kills cancer cells by disrupting the cell membrane and inducing apoptosis. The in vitro membrane selectivity toward cancer cells, such as A549 and MDA-MB-23, and human umbilical vein endothelial cells (HUVECs), normal cells, the penetrability assessment in the A549 3D multiple cell sphere model, and the in vivo tumor-tissue accumulation test in the A549 xenograft model indicated that HPRP-A1-iRGD exhibited significant increases in the selectivity toward membranes that highly express NRP-1, the penetration distance in 3D multiple cell spheres, and the accumulation in tumor tissues after intravenous injection, compared with HPRP-A1 alone. The mechanism of the enhanced targeting ability of HPRP-A1-iRGD was demonstrated by the pull-down assay and biolayer interferometry test, which indicated that the chimeric peptide could specifically bind to the neuropilin-1 protein with high affinity. We believe that chemical conjugation with iRGD to increase the specificity, penetration, and tumor-tissue accumulation of HPRP-A1 is an effective and promising approach for the targeted modification of peptides as anticancer therapeutics.

Background The commutability of electrolyte trueness verification materials (ETVs) and commercial general chemistry materials (GCs) was evaluated to investigate their suitability for use in an external quality assessment (EQA) program for serum sodium and potassium measurements. Methods Eighty fresh individual human samples (40 for sodium measurements and 40 for potassium measurements), six ETVs and three GCs were analyzed by five routine methods (validated methods) and by inductively coupled plasma mass spectrometry reference methods (comparative methods) for the determination of sodium and potassium. The commutability was analyzed according to Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol and difference in bias approach, respectively. The linearity, bias and imprecision of the routine methods were also assessed according to CLSI guidelines. Results According to EP14-A3 protocol, ETVs were commutable for all assays, and GCs were commutable for 3/5 assays for sodium. ETVs were commutable in most assays except Cobas C501, while GCs showed no commutability except in case of AU5821 for potassium. According to a difference in bias approach, the commutability of ETVs was inconclusive for most routine assays for both sodium and potassium, and GCs were inconclusive for sodium and non-commutable for potassium in most routine assays. The routine methods exhibited excellent linearities and precisions. The majority and minority of relative biases between the routine and reference methods were beyond the bias limits for sodium and potassium, respectively. Conclusions Superiority in the commutability of ETVs over GCs was observed among the sodium and potassium assays whichever evaluation approach was applied.

Conductive atomic force microscopy (C-AFM) is a powerful tool used in the microelectronics analysis by applying a certain bias voltage between the conducting probe and the sample and obtaining the electrical information of sample. In this work, the surface morphological information and current images of the lambda DNA (λ DNA) molecules with different distributions were obtained by C-AFM. The 1 and 10 ng μl−1 DNA solutions were dripped onto mica sheets for making randomly distributed DNA and DNA network samples, and another 1 ng μl−1 DNA sample was placed in a DC electric field with a voltage of 2 V before being dried for stretching the DNA sample. The results show that the current flowing through DNA networks was significantly higher than the stretched and random distribution of DNA in the experiment. The I–V curve of DNA networks was obtained by changing the bias voltage of C-AFM from −9 to 9 V. The currents flowing through stretched DNA at different pH values were studied. When the pH was 7, the current was the smallest, and the current was gradually increased as the solution became acidic or alkaline.

Large-scale and morphologically controlled self-assembled λ-DNA networks were successfully constructed by the synergistic effect of a DC electric field. The effect of DNA concentration, direction, and intensity of the electric field, even the modification of the mica surface using Mg

Large-scale and morphologically controlled self-assembled λ-DNA networks were successfully constructed by the synergistic effect of DC electric field. The effect of DNA concentration, direction and intensity of the electric field, even the modification of the mica surface using Mg2+ on the characteristics of the as-prepared DNA networks were investigated in detail by atomic force microscopy (AFM). It was found that the horizontal electric field was more advantageous to the formation of DNA networks with more regular structures. At the same concentration, the height of DNA network was not affected significantly by the intensity change of the horizontal electric field. The modification of Mg2+ on mica surface increased the aggregation of DNA molecules, which contributed to the morphological change of the DNA networks. Furthermore, DNA molecules were obviously stretched in both horizontal and vertical electric fields at low DNA concentrations.Statement of significanceThrough the synergistic effect of DC electric field, a series of large-scale and morphologically controlled self-assembled λ-DNA networks were successfully fabricated. We found that the horizontal electric field was more advantageous to the formation of DNA networks with more regular structures. At the same concentration of DNA solution, the height of DNA network was not affected significantly by the intensity change of the horizontal electric field. The modification of Mg2+ on mica surface increased the aggregation of DNA molecules, which contributed to the morphological change of the DNA networks. We suggest this study will promote the understanding on the preparation of controllable self-assembled λ-DNA networks and the application of DNA networks.

Hepatocellular carcinoma (HCC) is one of major leading causes of cancer death worldwide. As a traditional medicine, the anti-cancer function of ginseng is being growingly recognized and investigated. However, the effect of ginseng rust rot on human HCC is unknown yet. In this study, the HCC cells were treated with different parts of mountain cultivated ginseng rust rot and compared with human normal liver cells. The morphology, survival rate and β-actin expression of the cells were changed by introducing the ginseng epidermis during the incubation process. Notably, the results reveal that the ginseng epidermis can induce apoptosis by altering the morphologies of cells, indicating the practical implication for the HCC treatment and drug development.

Background Calcium is an important serum ion which is frequently assayed in clinical laboratories. Since quality assurance requires reference methods, the establishment of a candidate reference method for serum calcium measurement is important. Method An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were spiked gravimetrically with aluminum internal standard, digested with 69% ultrapure nitric acid and diluted to test concentration. Then the 44Ca/27Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators and the calibration function was established with bracketing method. Results The correlation coefficients between the measured 44Ca/27Al ratios and the analyte concentrations ratios were all > 0.9999. The coefficients of variation of the measurements were 0.27% and 0.16% for two spiked serum. The analytical recovery was 100.24%. The accuracy of the measurement was also verified through measurement of certified reference materials. Comparison with recognized reference method and international inter-laboratory comparisons gave satisfied results. Conclusion New ICP-MS method is specific, precise, simple, and low in cost, and may be used as a candidate reference method in the standardization of serum calcium measurement.

Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

Background Serum uric acid is a critical clinical indicator, and results without equivalence among laboratories cause troubles for disease diagnosis and patient management. External quality assessment (EQA) is a common tool for enhancing harmonization/standardization, therefore, the National Center for Clinical Laboratories in China has initiated a category 1 EQA for serum uric acid measurement since 2010 for evaluating its process of standardization. Methods Commutable EQA samples with target values assigned by reference measurement procedures were sent to participant laboratories. Both concentrations were measured 15 times in 3 days then means and intra-laboratory coefficient of variations (CVs) were reported. Biological variation criteria were used for analysis with CLIA88 criteria as a comparison. Results A total of 1250 laboratories participated in EQA programs from 2010 to 2017, pass rates calculated according to desirable specifications in biological variation database were on a rise overall and inter-laboratory mean bias and CVs were on a decrease. Homogeneous systems showed better inter-laboratory CVs and pass rates than heterogeneous systems. For the mostly used measurement systems; Abbott, Beckman, Roche Modular, Siemens and Hitachi showed desirable performances other than Roche Cobas, according to biological variation criteria. Conclusions Our study provides reliable information on the standardization of measurement procedures for serum uric acid for manufacturers and laboratories. Further improvements for standardization are still needed to make laboratories more patient-centered.

In this study, a peptide–peptide co-administration therapy between hybrid peptide kla-TAT and cationic anticancer peptide HPRP-A1 was designed to increase the anticancer activity of the combination peptides through synergistic effect. kla is a pro-apoptotic peptide which could induce rapid cancer cell apoptosis by disruption the mitochondrial membrane when internalized the cells. To enhance more kla peptides pass through cell membrane, a double improvement strategy was designed by chemically conjugation with cell penetration peptide TAT as well as co-administration with cationic membrane active peptide HPRP-A1, and the double anticancer mechanism of the kla-TAT peptide and HPRP-A1 including membrane disruption and apoptosis induction was verified through in vitro experiments. The CompuSyn synergism/antagonism analysis showed that kla-TAT acted synergistically with HPRP-A1 against a non-small cell lung cancer (NSCLC) A549 cell line. The anticancer activities of the two peptides were dramatically increased by co-administration, under the mechanism of cell membrane disruption, caspase-dependent apoptosis induction, as well as cyclin-D1 down-regulation based G1 phase arrest. We believe that the synergic therapeutic strategy would be a meaningful method for the anticancer peptides used in cancer treatment.

To improve the specificity and penetration of anticancer peptides against tumors, in this study, we examined the effects of co-administration of the membrane-active peptide HPRP-A1 and the tumor homing/penetrating peptide iRGD. iRGD peptide is widely recognized as an efficient cell membrane penetration peptide targeting to αvβ3 integrins and neuropilin-1 (NRP-1) receptors, which show high expression in many tumor cells. The anticancer activity, cancer specificity and penetration activity in vitro and in vivo of the co-administered peptides were examined on 2D monolayer cells, 3D multi-cellular spheroids (MCS) and xenograft nude mice. Co-administration of iRGD and HPRP-A1 exhibited stronger anticancer activity and tumor specificity against A549 non-small cell lung cancer cells with NRP-1 receptor overexpression compared with HPRP-A1 alone. A549 cells showed uptake of the peptide combination and destruction of the integrity of the cell membrane, as well as adherence to the mitochondrial net, resulting in induction of apoptosis by a caspase-dependent pathway. The iRGD peptide dramatically increased the penetration depth of HPRP-A1 on A549 MCS and anticancer efficacy in an A549 xenograft mouse model. Our results suggest that the co-administration strategy of anticancer and penetrating peptides could be a potential therapeutic approach for cancer treatment in clinical practice.

Highly stable giant supramolecular vesicles were constructed by hierarchical self-assembly of cucurbit[8]uril (CB[8])-based supra-amphiphiles for photoresponsive and targeted intracellular drug delivery. These smart vesicles can encapsulate the model drugs with high loading efficiencies and then release them by manipulating photoswitchable CB[8] heteroternary complexation to regulate the formation and dissociation of supra-amphiphiles that cause dramatic morphological changes of the assemblies to achieve remote optically controlled drug delivery. More importantly, the confocal microscopy analysis, cellular uptake experiment, and cell viability assay have shown that the giant vesicles are able to maintain the structural integrity and stability within actual cellular environments and exhibit obvious advantages for intracellular drug delivery such as low toxicity, easy surface modification for tumor-targeting selectivity, and rapid internalization into different human cancer cell lines. A synergistic mechanism that integrates multiple pathways including energy-dependent endocytosis, macropinocytosis, cholesterol-dependent endocytosis, and microtubule-related endocytosis was determined to facilitate the internalization process. Moreover, cytotoxicity experiments and flow cytometric analysis have demonstrated that the doxorubicin hydrochloride-loaded vesicles exhibited a significant therapeutic effect for tumor cells upon UV light irradiation, which makes the photoresponsive system more promising for potential applications in pharmaceutically relevant fields.

To enhance the anticancer activity, tumor penetration ability of the hybrid anticancer peptide, in this study, a TAT (RKKRRQRRR) peptide modified kla peptide (KLAKLAKKLAKLAK, with all D-amino acids), named kla-TAT, was co-administrated with the homing/penetrating peptide iRGD which could enhance the permeability of chemical drug in solid tumor and tumor vessel by co-administration. In this study, the nonsmall cell lung cancer A549 cell line with the iRGD targeting receptor neuropilin-1 high expression was selected to establish the 2D monolayer cell, 3D multiple cell spheroids, and xenograft mice model. The co-administration of iRGD strengthened the permeability of kla-TAT peptide against A549 2D and 3D sphere model with the penetration improvement property of iRGD; more importantly, co-administration with iRGD dramatically enhanced the accumulation of kla-TAT peptide in tumor tissue on the xenograft mice model with the homing property of iRGD. The co-administration of iRGD strategy confers targeting ability to the hybrid peptide kla-TAT. We believe the chemical conjugation plus co-administration approach may provide a promising way for cancer treatment in clinical practices.

Background: Hemoglobin A1c (HbA1c) measurement is of great value for the diagnosis and monitoring of diabetes. Many manufacturers have developed various experiments to determine the HbA1c concentration. However, the longitudinal use of these tests requires strict quality management. This study aimed to analyze the quality of HbA1c measurement systems in China using six sigma techniques to help improve their performances. Methods: A total of 135 laboratories were involved in this investigation in 2015. Bias values and coefficients of variation were collected from an HbA1c trueness verification external quality assessment program and an internal quality control program organized by the National Center of Clinical Laboratories in China. The sigma (σ) values and the quality goal index (QGI) were used to evaluate the performances of different groups, which were divided according to principles and instruments. Results: The majority of participants (88, 65.2%) were scored as “improvement needed (σ < 3)”, suggesting that the laboratories needed to improve their measurement performance. Only 8.2% (11/135) of the laboratories were scored as “world class (σ ≥ 6)”. Among all the 88 laboratories whose σ values were below 3, 52 (59.1%) and 23 (26.1%) laboratories needed to improve measurement precision (QGI 1.2), respectively; the remaining laboratories (13, 14.8%) needed to improve both measurement precision and trueness. In addition, 16.1% (5/31) and 15.0% (3/20) of the laboratories in “TOSOH” and “ARKRAY” groups, respectively, were scored as “world class”, whereas none of the laboratories in “BIO-RAD” group were scored as “world class”. Conclusions: This study indicated that, although participating laboratories were laboratories with better performance in China, the performances were still unsatisfactory. Actions should be taken to improve HbA1c measurement performance before we can include HbA1c assays in diabetes diagnosis in China.

Disulfiram (DSF), an FDA approved drug for the treatment of alcoholism, has shown its effectiveness against diverse cancer types. Thus, we developed a disulfiram-loaded scaffold using the electrospinning method to enhance the stability of DSF and to facilitate its appropriate distribution to tumor tissues. The drug release profile of the disulfiram-loaded scaffold was examined by high-performance liquid chromatography. We obtained mechanical and morphological characterizations of A549 cells treated with different scaffolds by various techniques to evaluate its antitumor properties. This work revealed that the cells after the treatment with the disulfiram-loaded scaffold exhibited a lower height and a larger elastic modulus compared with the untreated cells and those treated with the neat electrospun fibers. The changes were the indicators of cell apoptosis. Taken collectively, the results indicate that DSF was successfully incorporated into the electrospun fibers, and the disulfiram-loaded scaffold has great potential for inhibiting the regional recurrence of cancer.

Thyroid cancer is the most common tumor of the endocrine organs. Emerging studies have indicated the critical roles of microRNAs (miRs) in papillary thyroid cancer (PTC) formation and progression through function as tumor suppressors or oncogenes. The present study investigated the expression level and biological roles of miR-497 in PTC and its underlying mechanisms. It was demonstrated that the expression level of miR-497 was reduced in both PTC tissues and cell lines. Enforced expression of miR-497 suppressed PTC cell proliferation, migration and invasion. According to bioinformatics analysis, a luciferase reporter assay, reverse transcription-quantitative polymerase chain reaction and western blotting, RAC-γ serine/threonine-protein kinase (AKT3) was demonstrated to be the direct target gene of miR-497. In addition, AKT3 expression increased in PTC tissues and negatively correlated with miR-497 expression. Furthermore, downregulation of AKT3 also suppressed cell proliferation, migration and invasion of PTC, which had similar roles to miR-497 overexpression in PTC cells. Taken together, these results suggested that this newly identified miR-497/AKT3 signaling pathway may contribute to PTC occurrence and progression. These findings provide novel potential therapeutic targets for the therapy of PTC.

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.

Cyclin-dependent kinase BUR1/BUR2 appears to be the yeast ortholog of P-TEFb, which phosphorylates Ser2 of the RNA Pol II CTD, but the importance of BUR1/BUR2 in CTD phosphorylation is unclear. We show that BUR1/BUR2 is co-transcriptionally recruited to the 5′ end of ARG1 in a manner stimulated by interaction of the BUR1 C-terminus with CTD repeats phosphorylated on Ser5 by KIN28. Impairing BUR1/BUR2 function, or removing the CTD-interaction domain in BUR1, reduces Ser2 phosphorylation in bulk Pol II and eliminates the residual Ser2P in cells lacking the major Ser2 CTD kinase, CTK1. Impairing BUR1/BUR2 or CTK1 evokes a similar reduction of Ser2P in Pol II phosphorylated on Ser5, and in elongating Pol II near the ARG1 promoter. By contrast, CTK1 is responsible for the bulk of Ser2P in total Pol II and at promoter-distal sites. In addition to phosphorylating Ser2 near promoters, BUR1/BUR2 also stimulates Ser2P formation by CTK1 during transcription elongation.

Cancer is a great concern in the public health and development of novel therapeutic agent or strategy become urgently. Anticancer peptides (ACPs) have become promising anticancer drug candidate molecules due to their several extraordinary properties, such as small size, high activity, low immunogenicity, good biocompatibility, diversity of sequence and more modification sites for the functional molecules. However, the low stability and short half-life of peptides are the major barriers for the application. In this review, we focus on the methods of peptide design and modification to improve the stability, half-life time and specificity of ACPs, which including amino acid substitution, cyclization, hybridization, fragmentization, modification of C- and N-terminal of peptide by polymer or targeting molecules, etc.

Tigerinin-1R (Arg–Val–Cys–Ser–Ala–Ile–Pro–Leu–Pro–Ile–Cys–His–NH2), a cationic 12-mer peptide containing a disulfide bond extracted from frog skin secretions, lacks antibacterial activity, but has the ability to stimulate insulin release both in vitro and in vivo. To study the structure–function relationships of tigerinin-1R, we designed and synthesized five analogs, including tigerinin-cyclic, tigerinin-1R-L4, tigerinin-linear, [C3K]tigerinin-1R, and [C11K]tigerinin-1R. Tigerinin-1R promoted insulin secretion in a concentration-dependent manner in INS-1 cells without obvious cytotoxicity. At a concentration of 10−5 M, [C11K]tigerinin-1R exhibited the highest stimulation ability, suggesting that the positive charge at the C-terminus may contribute to the in vitro insulin-releasing activity of tigerinin-1R. Tigerinin-1R peptides stimulated insulin release in INS-1 cells through a universal mechanism that involves mobilization of intracellular calcium without disrupting the cell membrane. In vivo experiments showed that both tigerinin-1R and [C11K]tigerinin-1R improved glucose tolerance in overnight-fasted mice. Due to its structural stability, tigerinin-1R showed superior hypoglycemic activity to [C11K]tigerinin-1R, which suggested a critical role of the disulfide bonds. In addition, we also identified a protective effect of tigerinin-1R peptides in apoptosis induced by oxidative stress. These results further confirm the potential for the development of tigerinin-1R as an anti-diabetic therapeutic agent in clinical practice.

GCN2 stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating translation initiation factor 2. GCN2 is activated by binding of uncharged tRNA to a domain related to histidyl-tRNA synthetase (HisRS). The HisRS-like region contains two dimerization domains (HisRS-N and HisRS-C) required for GCN2 function in vivo but dispensable for dimerization by full-length GCN2. Residues corresponding to amino acids at the dimer interface of Escherichia coli HisRS were required for dimerization of recombinant HisRS-N and for tRNA binding by full-length GCN2, suggesting that HisRS-N dimerization promotes tRNA binding and kinase activation. HisRS-N also interacted with the protein kinase (PK) domain, and a deletion impairing this interaction destroyed GCN2 function without reducing tRNA binding; thus, HisRS-N-PK interaction appears to stimulate PK function. The C-terminal domain of GCN2 (C-term) interacted with the PK domain in a manner disrupted by an activating PK mutation (E803V). These results suggest that the C-term is an autoinhibitory domain, counteracted by tRNA binding. We conclude that multiple domain interactions, positive and negative, mediate the activation of GCN2 by uncharged tRNA.

The aim of this study was to compare the correlation and consistency of left ventricular ejection fraction (LVEF) obtained by ECG-gated myocardial perfusion SPET (GMPS) using the four formulas (R0-R3) in ECToolbox software and by equilibrium radionuclide ventriculography (ERNV), and determine the optimal diagnostic thresholds of the four formulas in a Chinese population. A hundred and three candidate donors (59 male and 44 female), including 38 patients with a history of myocardial infarction and 65 patients with suspected coronary heart disease, underwent both (99m)Tc-MIBI rest GMPS and technetium-99m red blood cells ((99m)Tc-RBC) ERNV within a week. The LVEF values calculated by ECToolbox R0, R1, R2 and R3 were compared with those obtained by ERNV. Using LVEF≥50% obtained by ERNV as the gold standard, the optimal diagnostic thresholds of the four formulas (R0-R3) were assessed by receiver operating characteristic (ROC) curves. Results showed that the mean LVEF value of ERNV was 54.6±17.5%, and the mean LVEF value of the four formulas was 64.1±15.7%, 56.3±15.1%, 69.9±17.9% and 56.3±13.6%, respectively, showing a significantly strong correlation between the results obtained by the two methods (r0.85, P0.001). All mean LVEF values obtained by the four formulas were higher than the mean LVEF value obtained by ERNV, and there was very significant difference between R0 and R2 results and the ERNV result (t=12.511 and 18.652, P0.001). Furthermore, there was significant difference between R1 and R3 results and the ERNV result (t=2.169 and 2.570, P0.05). Using ERNV LVEF≥50% as the normal diagnostic value, the optimal diagnostic threshold of R0∼R3 was 56.5%, 51.5%, 64.5% and 52.5%, respectively. There was a strong correlation between the LVEF values obtained by the four formulas in ECToolbox software and ERNV, but the numerical values of LVEF differed between the four formulas. In conclusion, A strong correlation was observed among R0, R1, R2 and R3 in the ECToolbox software when compared with ERNV and also between them for the assessment of LVEF. However, there were some differences in the numerical values of LVEF generated by the individual formulas, which must be taken into account in comparing clinical studies.

Paf1 complex (Paf1C) is a transcription elongation factor whose recruitment is stimulated by Spt5 and the CDKs Kin28 and Bur1, which phosphorylate the Pol II C-terminal domain (CTD) on Serines 2, 5, and 7. Bur1 promotes Paf1C recruitment by phosphorylating C-terminal repeats (CTRs) in Spt5, and we show that Kin28 enhances Spt5 phosphorylation by promoting Bur1 recruitment. It was unclear, however, whether CTD phosphorylation by Kin28 or Bur1 also stimulates Paf1C recruitment. We find that Paf1C and its Cdc73 subunit bind diphosphorylated CTD repeats (pCTD) and phosphorylated Spt5 CTRs (pCTRs) in vitro, and that cdc73 mutations eliminating both activities reduce Paf1C recruitment in vivo. Phosphomimetic (acidic) substitutions in the Spt5 CTR sustain high-level Paf1C recruitment in otherwise wild-type cells, but not following inactivation of Bur1 or Kin28. Furthermore, inactivating the pCTD/pCTR-interaction domain (PCID) in Cdc73 decreases Paf1C-dependent histone methylation in cells containing non-phosphorylatable Spt5 CTRs. These results identify an Spt5 pCTR-independent pathway of Paf1C recruitment requiring Kin28, Bur1, and the Cdc73 PCID. We propose that pCTD repeats and Spt5 pCTRs provide separate interaction surfaces that cooperate to ensure high-level Paf1C recruitment.

Methylation of histone H3 by Set1 and Set2 is required for deacetylation of nucleosomes in coding regions by histone deacetylase complexes (HDACs) Set3C and Rpd3C(S), respectively. We report that Set3C and Rpd3C(S) are co-transcriptionally recruited in the absence of Set1 and Set2, but in a manner stimulated by Pol II CTD kinase Cdk7/Kin28. Consistently, Rpd3C(S) and Set3C interact with Ser5-phosphorylated Pol II and histones in extracts, but only the histone interactions require H3 methylation. Moreover, reconstituted Rpd3C(S) binds specifically to Ser5-phosphorylated CTD peptides in vitro. Hence, whereas interaction with methylated H3 residues is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated CTD. We further demonstrate that Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating histones and suppressing co-transcriptional histone eviction. A strong correlation between increased acetylation and lower histone occupancy in HDA mutants implies that histone acetylation is a key determinant of nucleosome eviction.

The calculation and exhibition of risk management techniques will be more efficient and flexible by business intelligence(BI) tools. However, there are few researches on BI for securities companies' risk management(SCRM). After reviewing the business and technique problems, this paper analyzed the BI system framework of SCRM and discussed key issues of BI system construction. It also addressed the adaptive BI system structure of SCRM, which could flexibly adapt system parameters to the changing data.

Nuclear cap binding complex (CBC) is recruited cotranscriptionally and stimulates spliceosome assembly on nascent mRNAs; however, its possible functions in regulating transcription elongation or termination were not well understood. We show that, while CBC appears to be dispensable for normal rates and processivity of elongation by RNA polymerase II (Pol II), it plays a direct role in preventing polyadenylation at weak termination sites. Similarly to Npl3p, with which it interacts, CBC suppresses the weak terminator of the gal10-Delta56 mutant allele by impeding recruitment of termination factors Pcf11p and Rna15p (subunits of cleavage factor IA [CF IA]) and does so without influencing Npl3p occupancy at the termination site. Importantly, deletion of CBC subunits or NPL3 also increases termination at a naturally occurring weak poly(A) site in the RNA14 coding sequences. We also show that CBC is most likely recruited directly to the cap of nascent transcripts rather than interacting first with transcriptional activators or the phosphorylated C-terminal domain of Pol II. Thus, our findings illuminate the mechanism of CBC recruitment and extend its function in Saccharomyces cerevisiae beyond mRNA splicing and degradation of aberrant nuclear mRNAs to include regulation of CF IA recruitment at poly(A) selection sites.

The Paf1 complex (Paf1C) interacts with RNA polymerase II (Pol II) and promotes histone methylation of transcribed coding sequences, but the mechanism of Paf1C recruitment is unknown. We show that Paf1C is not recruited directly by the activator Gcn4p but is dependent on preinitiation complex assembly and Ser5 carboxy-terminal domain phosphorylation for optimal association with ARG1 coding sequences. Importantly, Spt4p is required for Paf1C occupancy at ARG1 (and other genes) and for Paf1C association with Ser5-phosphorylated Pol II in cell extracts, whereas Spt4p-Pol II association is independent of Paf1C. Since spt4Delta does not reduce levels of Pol II at ARG1, Ser5 phosphorylation, or Paf1C expression, it appears that Spt4p (or its partner in DSIF, Spt5p) provides a platform on Pol II for recruiting Paf1C following Ser5 phosphorylation and promoter clearance. spt4Delta reduces trimethylation of Lys4 on histone H3, demonstrating a new role for yeast DSIF in promoting a Paf1C-dependent function in elongation.

Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.

Wild-type transcriptional activation by Gcn4p is dependent on multiple coactivators, including SAGA, SWI/SNF, Srb mediator, CCR4-NOT, and RSC, which are all recruited by Gcn4p to its target promoters in vivo. It was not known whether these coactivators are required for assembly of the preinitiation complex (PIC) or for subsequent steps in the initiation or elongation phase of transcription. We find that mutations in subunits of these coactivators reduce the recruitment of TATA binding protein (TBP) and RNA polymerase II (Pol II) by Gcn4p at ARG1, ARG4, and SNZ1, implicating all five coactivators in PIC assembly at Gcn4p target genes. Recruitment of Pol II at SNZ1 and ARG1 was eliminated by mutations in TBP or by deletion of the TATA box, indicating that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However, several mutations in SAGA subunits and deletion of SRB10 had a greater impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and Srb mediator can promote Pol II binding independently of their stimulatory effects on TBP recruitment. Our results reveal an unexpected complexity in the cofactor requirements for the enhancement of PIC assembly by a single activator protein.

The protein kinase GCN2 is activated in amino acid-starved cells on binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-related domain. We isolated two point mutations in the protein kinase (PK) domain, R794G and F842L, that permit strong kinase activity in the absence of tRNA binding. These mutations also bypass the requirement for ribosome binding, dimerization, and association with the GCN1/GCN20 regulatory complex, suggesting that all of these functions facilitate tRNA binding to wild-type GCN2. While the isolated wild-type PK domain was completely inert, the mutant PK was highly active in vivo and in vitro. These results identify an inhibitory structure intrinsic to the PK domain that must be overcome on tRNA binding by interactions with a regulatory region, most likely the N terminus of the HisRS segment. As Arg 794 and Phe 842 are predicted to lie close to one another and to the active site, they may participate directly in misaligning active site residues. Autophosphorylation of the activation loop was stimulated byR794G and F842L, and the autophosphorylation sites remained critical for GCN2 function in the presence of these mutations. Our results imply a two-step activation mechanism involving distinct conformational changes in the PK domain.