Sq-2, a biotinylated annonaceous acetogenin, induces apoptosis, autophagy and S-phase arrest by activating the MAPK pathway in breast cancer cells.

Squamocin, an annonaceous acetogenin isolated from plants in the Annonaceae family, has antitumour activity. In this study, we report that Sq-2, a biotinylated squamocin monomer, has a favorable antitumour effect on MDA-MB-231 and SKBR3 breast cancer cells in vitro. MTT assays show that Sq-2 has a better antitumour effect on MDA-MB-231 cells than Sq-5 and Sq-6. Furthermore, RNA-Seq and KEGG enrichment analyses reveal that Sq-2 activates the MAPK signaling pathway, and results of western blot analysis demonstrate that Sq-2 activates the JNK and p38 pathways in MDA-MB-231 and SKBR3 cells. Flow cytometry and western blot analysis reveal that Sq-2 induces cell apoptosis by increasing the expressions of cleaved Caspase-3 and cleaved PARP as well as the ratio of Bax/Bcl-2. Inhibition of the Caspase family by Z-VAD-FMK attenuates the viability of MDA-MB-231 cells, indicating that Sq-2 induces apoptosis in a Caspase-dependent manner. Additionally, pretreatment with the p38 inhibitor SB203580 or JNK inhibitor SP600125 partially reverses the increase in the apoptosis rate and decrease in cell viability prompted by Sq-2. Furthermore, Sq-2 treatment decreases the expression level of CyclinD1 and increases the expression levels of p21, p27, CyclinA1, and CDK2, causing S-phase arrest in MDA-MB-231 and SKBR3 cells. Further study indicates that Sq-2 stimulates autophagy in MDA-MB-231 and SKBR3 cells, and inhibition of autophagy by bafilomycin A1 increases cell viability and promotes cell survival. Sq-2, a novel biotin-squamocin compound, shows a significant inhibitory effect on the propagation of SKBR3 and MDA-MB-231 breast cancer cells. Furthermore, Sq-2 treatment not only induces S-phase arrest and activates the JNK and p38 pathways to trigger apoptosis but also causes autophagy to promote apoptosis in MDA-MB-231 and SKBR3 cells.

Direct and Rapid Identification of Vibrio Cholerae Serogroup and Toxigenicity by a Novel Multiplex Real-Time Assay.

Molecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens and more targets in one turnaround time simultaneously. In this study, we utilized nucleic acid extraction-free, direct RT-PCR and high-speed amplification to develop a novel multiplex assay, a quadplex direct one-tube real-time RT-PCR assay, for rapid detection of the serogroup and cholera toxin toxigenicity of Vibrio cholerae targeting the epsM, ctxA, rfb-O1, and rfb-O139 genes. Performance of the multiplex assay was evaluated by comparison with the monoplex real-time PCR assay according to the China Cholera Prevention Manual. Detection data from clinical specimens showed that the new assay had good diagnostic sensitivities for epsM (100%, n = 301), ctxA (100%, n = 125), rfb-O1 (100%, n = 85), and rfb-O139 (97.87%, n = 49). Analysis of the analytical sensitivities with serial dilutions of positive standards showed that the detection limits of the new assay for Vibrio cholerae epsM,ctxA,rfb-O1, and rfb-O139 were up to 200, 590, 115, and 1052 copies per mL lower than the monoplex real-time PCR (910, 345, and 1616 copies/mL respectively, for ctxA,rfb-O1, and rfb-O139). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for Vibrio cholerae, especially suitable for rapid identification and screening detection of mass specimens.

Biomechanical responses of human lumbar spine and pelvis according to the Roussouly classification.

BACKGROUND: Few studies have analyzed the different biomechanical properties of the lumbar with various morphological parameters, which play an important role in injury and degeneration. This study aims to preliminarily investigate biomechanical characteristics of the spine with different sagittal alignment morphotypes by using finite element (FE) simulation and in-vitro testing. METHODS: According to the lumbar-pelvic radiographic parameters of the Chinese population, the parametric FE models (L1-S1-pelvis) of Roussouly's type (1-4) were validated and developed based on the in-vitro biomechanical testing. A pure moment of 7.5 Nm was applied in the three anatomical planes to simulate the physiological activities of flexion, extension, left-right lateral bending and left-right axial rotation. RESULTS: The sagittal configuration of four Roussouly's type models had a strong effect on the biomechanical responses in flexion and extension. The apex of the lumbar lordosis is a critical position where the segment has the lowest range of motion among all the models. In flexion-extension, type 3 and 4 models with a good lordosis shape had a more uniform rotation distribution at each motor function segment, however, type 1 and 2 models with a straighter spine had a larger proportion of rotation at the L5-S1 level. In addition, type 1 and 2 models had higher intradiscal pressures (IDPs) at the L4-5 segment in flexion, while type 4 model had larger matrix and fiber stresses at the L5-S1 segment in extension. CONCLUSION: The well-marched lordotic type 3 lumbar had greater stability, however, a straighter spine (type 1 and 2) had poor balance and load-bearing capacity. The hypolordotic type 4 model showed larger annulus fiber stress. Therefore, the sagittal alignment of Roussouly's type models had different kinetic and biomechanical responses under various loading conditions, leading to different clinical manifestations of the lumbar disease.

The Efficient and Convenient Extracting Uranium from Water by a Uranyl-Ion Affine Microgel Container.

Uranium is an indispensable part of the nuclear industry that benefits us, but its consequent pollution of water bodies also makes a far-reaching impact on human society. The rapid, efficient and convenient extraction of uranium from water is to be a top priority. Thanks to the super hydrophilic and fast adsorption rate of microgel, it has been the ideal adsorbent in water; however, it was too difficult to recover the microgel after adsorption, which limited its practical applications. Here, we developed a uranyl-ion affine and recyclable microgel container that has not only the rapid swelling rate of microgel particles but also allows the detection of the adsorption saturation process by the naked eye.

Development and Application of a Multiplex Fluorescent PCR for Shigella Detection and Species Identification.

This study was to develop a multiplex fluorescent PCR for Shigella detection and species identification. Five primer pairs for Shigella detection and species identification were designed by Primer Premier 5.0. The multiplex fluorescent PCR was optimized by varying single parameter while other parameters were maintained. The multiplex fluorescent PCR assay could correctly detect Shigella and identify four Shigella species with a detection limits of 10 pg genomic DNA per reaction. Testing different strains and clinical samples confirmed the sensitivity and specificity of the multiplex fluorescent PCR. The newly developed multiplex fluorescent PCR assay is simple, sensitive and specific for Shigella detection and species identification. It has a potential to be used in routine Shigella detection and species identification in clinical laboratories.

Analysis of Vulnerability on Weighted Power Networks under Line Breakdowns.

Vulnerability is a major concern for power networks. Malicious attacks have the potential to trigger cascading failures and large blackouts. The robustness of power networks against line failure has been of interest in the past several years. However, this scenario cannot cover weighted situations in the real world. This paper investigates the vulnerability of weighted power networks. Firstly, we propose a more practical capacity model to investigate the cascading failure of weighted power networks under different attack strategies. Results show that the smaller threshold of the capacity parameter can enhance the vulnerability of weighted power networks. Furthermore, a weighted electrical cyber-physical interdependent network is developed to study the vulnerability and failure dynamics of the entire power network. We perform simulations in the IEEE 118 Bus case to evaluate the vulnerability under various coupling schemes and different attack strategies. Simulation results show that heavier loads increase the likelihood of blackouts and that different coupling strategies play a crucial role in the cascading failure performance.

Apoptosis-promoting properties of miR-3074-5p in MC3T3-E1 cells under iron overload conditions.

BACKGROUND: Iron overload can promote the development of osteoporosis by inducing apoptosis in osteoblasts. However, the mechanism by which miRNAs regulate apoptosis in osteoblasts under iron overload has not been elucidated. METHOD: The miRNA expression profile in MC3T3-E1 cells under iron overload was detected by next generation sequencing. qRT-PCR was used to determine the expression of miR-3074-5p in MC3T3-E1 cells under iron overload. The proliferation of MC3T3-E1 cells was tested using CCK-8 assays, and apoptosis was measured using flow cytometry. The miRanda and TargetScan databases were used to predict the target genes of miR-3074-5p. Interaction between miR-3074-5p and the potential target gene was validated by qRT-PCR, luciferase reporter assay and western blotting. RESULTS: We found that iron overload decreased the cell viability and induced apoptosis of MC3T3-E1 cells. The results of next generation sequencing analysis showed that miR-3074-5p expression was significantly increased in MC3T3-E1 cells under iron overload conditions, which was confirmed by further experiments. The inhibition of miR-3074-5p attenuated the apoptosis of iron-overloaded MC3T3-E1 cells. Furthermore, the expression of Smad4 was decreased and was inversely correlated with miR-3074-5p expression, and overexpression of Smad4 partially reversed the viability inhibition of iron-overloaded MC3T3-E1 cells by relieving the suppression of ERK, AKT, and Stat3 phosphorylation, suggesting its regulatory role in the viability inhibition of iron-overloaded MC3T3-E1 cells. The luciferase reporter assay results showed that Smad4 was the target gene of miR-3074-5p. CONCLUSION: miR-3074-5p functions as an apoptosis promoter in iron-overloaded MC3T3-E1 cells by directly targeting Smad4.

Dynamic Colloidal Photonic Crystal Hydrogels with Self-Recovery and Injectability.

Simulation of self-recovery and diversity of natural photonic crystal (PC) structures remain great challenges for artificial PC materials. Motivated by the dynamic characteristics of PC nanostructures, here, we present a new strategy for the design of hydrogel-based artificial PC materials with reversible interactions in the periodic nanostructures. The dynamic PC hydrogels, derived from self-assembled microgel colloidal crystals, were tactfully constructed by reversible crosslinking of adjacent microgels in the ordered structure via phenylboronate covalent chemistry. As proof of concept, three types of dynamic colloidal PC hydrogels with different structural colors were prepared. All the hydrogels showed perfect self-healing ability against physical damage. Moreover, dynamic crosslinking within the microgel crystals enabled shear-thinning injection of the PC hydrogels through a syringe (indicating injectability or printability), followed by rapid recovery of the structural colors. In short, in addition to the great significance in biomimicry of self-healing function of natural PC materials, our work provides a facile strategy for the construction of diversified artificial PC materials for different applications such as chem-/biosensing, counterfeit prevention, optical display, and energy conversion.

Active Monomer RTR-1 Derived from the Root of Rhodomyrtus t omentosa Induces Apoptosis in Gastric Carcinoma Cells by Inducing ER Stress and Inhibiting the STAT3 Signaling Pathway.

PURPOSE: Rhodomyrtus tomentosa, a flowering plant from the Myrtaceae family, is considered  an antitumour substance with versatile biological and pharmacological activities. RTR-1 is an active monomer purified from the root of Rhodomyrtus tomentosa. However, the detail of mechanism involving in RTR-1 anti-cancer activity remains to be elucidated, and the effect on gastric cancer cells is unknown. METHODS: Cell proliferation was determined by MTT and clone formation assay. The effect of RTR-1 on cell cycle distribution and apoptosis was analysed utilizing flow cytometry, respectively. Moreover, Western blotting was used to detect the expression of cell cycle- and apoptosis-related protein. RESULTS: Based on MTT and clone formation assay, we noticed that RTR-1 inhibited the proliferation of gastric carcinoma (BGC823 and SGC7901) cells in a dose- and time-dependent manner. Furthermore, the results of flow cytometry and Western blotting showed that RTR-1 induced cell cycle arrest in the G2/M phase through the ATM-Chk2-p53-p21 signaling pathway and induced cell apoptosis by inhibiting the signal transducers and activators of transcription 3 (STAT3) pathway and activating the endoplasmic reticulum stress (ER stress) pathway. CONCLUSION: Taken together, these results demonstrate that RTR-1 induces cell cycle arrest and promotes apoptosis in gastric carcinoma, indicating its potential application for gastric cancer therapy.

[Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3].

Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.

Mussel-Derived, Cancer-Targeting Peptide as pH-Sensitive Prodrug Nanocarrier.

In this work, we prepared a novel cancer chemotherapeutic nanocarrier through the self-assembly of a mussel-derived, cancer-targeting peptide with a pH-sensitive conjugation of antitumor drugs. The biomimetic peptide was designed with a fluorescent molecule fluorescein isothiocyanate for imaging, a RGD sequence for cancer-targeting and tetravalent catechol groups for dynamic conjugation of the antitumor drug bortezomib via pH-cleavable boronic acid-catechol esters. Our study demonstrated that the peptide-based prodrug nanocarrier dramatically the enhanced specific cellular uptake and cytotoxicity toward human breast cancer cells in vitro in comparison with free drug and nontargeting control nanoparticles. Likewise, the prodrug nanocarrier showed improved therapeutic efficacy and low systematic toxicity in vivo. Considering highly biomimetic nature of the peptide-based nanocarriers, rapid drug release from the dynamically conjugated prodrugs, and convenience of introducing cancer-targeting activity onto this nanosystem, we believe our work would provide new ideas for the development of intelligent and biocompatible drug delivery systems to improve the chemotherapy efficacy in clinic. Furthermore, the pH-sensitive drug conjugation mechanism on peptide-based nanocarriers would provide a hint for the exploitation of dynamic prodrug strategies and the development of highly biocompatible nanocarriers using biogenic materials, e.g., the proteinogenic nanomaterials decorated with drugs through dynamic covalent chemistry.

A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus.

Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.

A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses.

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5-25 viral RNA copies per µl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/µl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/µl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

Development and Application of A Duplex Real-Time RT-PCR Assay for Simultaneous Detection of Coxsackievirus A6 and Coxsackievirus A10.

Hand, foot and mouth disease (HFMD) is a serious public health problem. Generally, it is considered that HFMD is mainly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). Nevertheless, the incidence of HFMD caused by coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) has increased significantly and CVA6 and CVA10 have become major causes of HFMD epidemic. This study develops a duplex real-time reverse transcript polymerase chain reaction (RT-PCR) assay for simultaneous detection of CVA 6 and CVA 10. The specificity, sensitivity, and reproducibility of this assay were analyzed. No cross-reactions with other viruses or false positives were observed. The detection limit of this assay was as low as 11.935 copies for CVA6 and 17.591 copies for CVA10 per reaction (concentration giving a positive duplex real-time RT-PCR result in 95% of samples). The coefficients of variation of the intra- and inter-assay reproducibility for CVA 6 and CVA 10 were both lower than 2%. Our results showed that this duplex real-time RT-PCR assay was a simple, rapid, and cost-effective method for simultaneous identification of CVA6 and CVA10.

Dynamic quantification of avian influenza H7N9(A) virus in a human infection during clinical treatment using droplet digital PCR.

This study involved a human infection with avian influenza H7N9(A) virus in Zhejiang province, the first one after implementing the closure measures of living poultry markets in China. The clinical symptoms, epidemiological and virological characteristics of the case were described briefly, and as the emphasis, H7N9 virus was detected quantitatively and continuously from the collected samples in 10 different periods of the patient's treatment in order to reveal changes of viral load in patient's body during the treatment. This study first used reverse-transcription droplet digital PCR (RT-ddPCR) assays to monitor viral load dynamically for human H7N9 infection, synchronously performing real-time RT-PCR as a reference technology to obtain more comprehensive data for comparison. Our results indicated that RT-ddPCR compared to real-time RT-PCR is more sensitive and accurate for quantifying H7N9 viral load without the use of standard curves. Furthermore it can provide reference data for clinical policies including infectivity judgement, ward transferring and therapy adjustment for the patient during treatment.

Multiplex real-time PCR assay for detection of pathogenic Vibrio parahaemolyticus strains.

Foodborne disease caused by pathogenic Vibrio parahaemolyticus has become a serious public health problem in many countries. Rapid diagnosis and the identification of pathogenic V. parahaemolyticus are very important in the context of public health. In this study, an EvaGreen-based multiplex real-time PCR assay was established for the detection of pathogenic V. parahaemolyticus. This assay targeted three genetic markers of V. parahaemolyticus (species-specific gene toxR and virulence genes tdh and trh). The assay could unambiguously identify pathogenic V. parahaemolyticus with a minimum detection limit of 1.4 pg genomic DNA per reaction (concentration giving a positive multiplex real-time PCR result in 95% of samples). The specificity of the assay was evaluated using 72 strains of V. parahaemolyticus and other bacteria. A validation of the assay with clinical samples confirmed its sensitivity and specificity. Our data suggest the newly established multiplex real-time PCR assay is practical, cost-effective, specific, sensitive and capable of high-throughput detection of pathogenic V. parahaemolyticus.

Inhibition of macrophage autophagy induced by Salmonella enterica serovar typhi plasmid.

pR(ST98), a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi), is involved in bacterial multidrug-resistance and virulence, however, its exact contributions to bacterial pathogenesis are still not fully understood. To investigate whether pR(ST98) exhibits potential to mediate macrophage autophagy and apoptosis, murine macrophage-like cell line (J774A.1) was infected with wild type strain (S. typhi-WT), mutant strain (S. typhi-DeltapR(ST98)) and complement of S. typhi-DeltapR(ST98) (S. typhi-c-pR(ST98)). Results revealed that S. typhi harboring pR(ST98) decreased the number of autophagy vacuoles of macrophages as well as the expression of Beclin 1 and LC3-II at the early stage of infection; apoptosis rate of macrophages infected with S. typhi-DeltapR(ST98) was lower than that infected with S. typhi-WT or S. typhi-c-pR(ST98). The survival rate of intracellular bacteria carrying pR(ST98) was much higher than that of plasmid free strain. After intervention with autophagy agonist rapamycin, apoptosis rate of the cells infected with S. typhi containing pR(ST98) and intracellular bacterial growth decreased. Our study suggested that pR(ST98) could inhibit autophagy and induce cell apoptosis for the host bacterial survival and proliferation.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV.

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).

Salmonella enterica serovar Typhi plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy.

OBJECTIVES: Plasmid pR ST98 is a hybrid resistance-virulence plasmid isolated from Salmonella enterica serovar Typhi (S. typhi). Previous studies demonstrated that pR ST98 could enhance the virulence of its host bacteria. However, the mechanism of pR ST98-increased bacterial virulence is still not fully elucidated. This study was designed to gain further insight into the roles of pR ST98 in host responses. METHODS: Human-derived macrophage-like cell line THP-1 was infected with wild-type (ST8), pR ST98-deletion (ST8-ΔpR ST98), and complemented (ST8-c-pR ST98) S. typhi strains. Macrophage autophagy was performed by extracting the membrane-unbound LC3-I protein from cells, followed by flow cytometric detection of the membrane-associated fraction of LC3-II. Intracellular bacterial growth was determined by colony-forming units (cfu) assay. Macrophage cell death was measured by flow cytometry after propidium iodide (PI) staining. Autophagy activator rapamycin (RAPA) was added to the medium 2 h before infection to investigate the effect of autophagy on intracellular bacterial growth and macrophage cell death after S. typhi infection. RESULTS: Plasmid pR ST98 suppressed autophagy in infected macrophages and enhanced intracellular bacterial growth and S. typhi-induced macrophage cell death. Pretreatment with RAPA effectively restricted intracellular bacterial growth of ST8 and ST8-c-pR ST98, and alleviated ST8 and ST8-c-pR ST98-induced macrophage cell death, but had no significant effect on ST8-ΔpR ST98. CONCLUSIONS: Plasmid pR ST98 enhances intracellular bacterial growth and S. typhi-induced macrophage cell death by suppressing autophagy.