Development and evaluation of human T‐cell leukemia virus‐1 and ‐2 multiplex quantitative PCR

The diagnosis of human T ‐cell leukemia virus type 1 (HTLV‐1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV‐1 and/or HTLV‐2 are used to confirm infection with HTLV‐1 and/or HTLV‐2 and are also used for the follow‐up of HTLV‐1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV‐1 and HTLV‐2, a multiplex quantitative polymerase chain reaction (qPCR) by large‐scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV‐1 provirus per 10⁵ cells. Moreover, HTLV‐1 provirus could be detected in 97.2% (205 of 211) of HTLV‐1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV‐2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV‐1 and HTLV‐2 provirus with extremely high sensitivity.     

Refined structures of mouse P‐glycoprotein

The recently determined C. elegans P‐glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single‐wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Å resolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug‐free structure was refined to 3.8 Å resolution with a 9.4 and 8.1% decrease in R_work and R_free, respectively, (R_work = 21.2%, R_free = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ～95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N‐terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Å from their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.     

P‐bodies and their functions during mRNA cell cycle: Mini‐review

P‐bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P‐bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P‐bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P‐bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P‐bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini‐review, we focus on the structure of P‐bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression. Copyright © 2012 John Wiley & Sons, Ltd.     

Down‐regulation of c‐fos by shRNA sensitizes adriamycin‐resistant MCF‐7/ADR cells to chemotherapeutic agents via P‐glycoprotein inhibition and apoptosis augmentation

Multidrug resistance (MDR) is a major hurdle in the treatment of cancer. Research indicated that the main mechanisms of most cancers included so‐called “pump” (P‐glycoprotein, P‐gp) and “non‐pump” (apoptosis) resistance. Identification of novel signaling molecules associated with both P‐gp and apoptosis will facilitate the development of more effective strategies to overcome MDR in tumor cells. Since the proto‐oncogene c‐fos has been implicated in cell adaptation to environmental changes, we analyzed its role in mediating “pump” and “non‐pump” resistance in MCF‐7/ADR, an adriamycin (ADR)‐selected human breast cancer cell line with the MDR phenotype. Elevated expression of c‐fos in MCF‐7/ADR cells and induction of c‐fos by ADR in the parental drug‐sensitive MCF‐7 cells suggested a link between c‐fos and MDR phenotype. Down‐regulation of c‐fos expression via shRNA resulted in sensitization of MCF‐7/ADR cells to chemotherapeutic agents, including both P‐gp and non‐P‐gp substrates. Further results proved that c‐fos down‐regulation in MCF‐7/ADR cells resulted in decreased P‐gp expression and activity, enhanced apoptosis, and altered expression of apoptosis‐associated proteins (i.e., Bax, Bcl‐2, p53, and PUMA). All above facts indicate that c‐fos is involved in both P‐gp‐ and anti‐apoptosis‐mediated MDR of MCF‐7/ADR cells. Based on these results, we propose that c‐fos may represent a potential molecular target for resistant cancer therapy, and suppressing c‐fos gene expression may therefore be an effective means to temper breast cancer cell's MDR to cytotoxic chemotherapy. J. Cell. Biochem. 114: 1890–1900, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of P‐Glycoprotein‐Induced Multidrug Resistance by a Clerodane‐Type Diterpenoid from Sindora sumatrana

The aim of the present study was to investigate the effects of di‐ and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P‐glycoprotein (P‐gp) function in an adriamycin‐resistant human breast cancer cell line, MCF‐7/ADR. Over‐expression of P‐gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di‐ and sesquiterpenoids extracted from S. sumatrana, (+)‐7β‐acetoxy‐15,16‐epoxycleroda‐3,13(16),14‐trien‐18‐oic acid ( 1 ) showed a strong P‐gp inhibitory effect, as great as that of verapamil, a representative P‐gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC₅₀ value for daunomycin. These results suggest that compound 1 inhibits the functioning of P‐gp and, therefore, can be developed as an MDR‐reversing agent.     

Iron metabolism markers and haptoglobin phenotypes in susceptibility to HSV‐1 or/and HSV‐2 lesion relapses

Different haptoglobin (Hp) phenotypes play a role in several pathologic processes including infectious diseases. In order to evaluate the role of iron storage and metabolism in susceptibility to herpetic manifestations, we studied the frequency of the Hp phenotypes and iron metabolism in patients affected by H. Simplex virus 1 or 2 (HSV‐1 or HSV‐2), compared with controls. Hp phenotype and iron metabolism were determined in 100 patients with recurrent HSV‐1 or HSV‐2 manifestations during the relapses, and in 110 healthy subjects. The frequencies of the three Hp phenotypes in the patient group compared to the control group were 18% versus 14.5% p = NS for Hp 1.1, 25% versus 40% p = 0.03 for Hp 2.2 and 57% versus 45.5% p = NS for Hp 2.1. All iron metabolism parameters tested showed significant differences between patients and controls; haemoglobin (Hb), ferritin, and serum iron were lower, while transferrin was higher in the patients than in controls. Reductions in iron availability may be a risk factor for relapsing lesions of HSV‐1 or HSV‐2. Hp 2.2 phenotype may offer some protection against the recurrence of Herpes labialis or genitalis manifestations. Copyright © 2010 John Wiley & Sons, Ltd.     

Metal–Organophosphine Framework‐Derived N,P‐Codoped Carbon‐Confined Cu3P Nanopaticles for Superb Na‐Ion Storage

Metal–organic framework derived approaches are emerging as a viable way to design carbon‐confined transitional metal phosphides (TMPs@C) for energy storage and conversion. However, their preparation generally involves a phosphorization using a large amount of additional P sources, which inevitably releases flammable, poisonous PH₃. Therefore, developing an efficient strategy for eco‐friendly synthesis of TMPs@C is full of challenges. Here, a metal–organophosphine framework (MOPF) derived strategy is developed to allow an eco‐friendly design of TMPs@C without an additional P source, avoiding release of PH₃. To illustrate this strategy, 1,3,5‐triaza‐7‐phosphaadamantane (PTA) ligands and Cu(NO₃)₂ metal centers are employed to construct Cu/PTA‐MOPFs nanosheets. Cu/PTA‐MOPFs can be directly converted to carbon‐confined Cu₃P nanoparticles by annealing. Benefiting from high heteroatom content in PTA, a high doping content of 3.92 at% N and 8.26 at% P can also be achieved in the carbon matrix. As a proof‐of‐concept application, N,P‐codoped carbon‐confined Cu₃P nanoparticles as anodes for Na‐ion storage exhibit a high initial reversible capacity of 332 mA h g^?1 at 50 mA g^?1, and superb rate and cyclic performance. Due to rich coordination modes of organophosphine, MOPFs are expected to become a promising molecular platform for design of various heteroatom‐doped TMPs@C for energy storage and conversion.     

Hepatic 31P‐magnetic resonance spectroscopy identified the impact of melatonin‐pretreated mitochondria in acute liver ischaemia‐reperfusion injury

Acute liver ischaemia‐reperfusion injury (IRI), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. However, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. This study tested the hypothesis that ³¹P‐magnetic resonance spectroscopy (³¹P‐MRS) findings could provide reliable living images to accurately identify the degree of acute liver IRI and melatonin‐pretreated mitochondria was an innovative treatment for protecting the liver from IRI in rat. Adult male SD rats were categorized into group 1 (sham‐operated control), group 2 (IRI only) and group 3 (IRI + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). By the end of study period at 72 hours, ³¹P‐MRS showed that, as compared with group 1, the hepatic levels of ATP and NADH were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. The liver protein expressions of mitochondrial‐electron‐transport‐chain complexes and mitochondrial integrity exhibited an identical pattern to ³¹P‐MRS finding. The protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial‐damaged biomarkers displayed an opposite finding of ³¹P‐MRS, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. Microscopic findings showed that the fibrotic area/liver injury score and inflammatory and DNA‐damaged biomarkers exhibited an identical pattern of cellular stress signalling. Melatonin‐pretreated mitochondria effectively protected liver against IRI and ³¹P‐MRS was a reliable tool for measuring the mitochondrial/ATP consumption in living animals.     

Probing the activity of a recombinant Zn2+‐transporting P‐type ATPase

P‐type ATPase proteins maintain cellular homeostasis and uphold critical concentration gradients by ATP‐driven ion transport across biological membranes. Characterization of single‐cycle dynamics by time‐resolved X‐ray scattering techniques in solution could resolve structural intermediates not amendable to for example crystallization or cryo‐electron microscopy sample preparation. To pave way for such time‐resolved experiments, we used biochemical activity measurements, Attenuated Total Reflectance (ATR) and time‐dependent Fourier‐Transform Infra‐Red (FTIR) spectroscopy to identify optimal conditions for activating a Zn²⁺‐transporting Type‐I ATPase from Shigella sonnei (ssZntA) at high protein concentration using caged ATP. The highest total activity was observed at a protein concentration of 25 mg/mL, at 310 K, pH 7, and required the presence of 20% (v/v) glycerol as stabilizing agent. Neither the presence of caged ATP nor increasing lipid‐to‐protein ratio affected the hydrolysis activity significantly. This work also paves way for characterization of recombinant metal‐transporting (Type‐I) ATPase mutants with medical relevance.     

Anti‐HIV‐1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication

The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.     

Layered Na‐Ion Cathodes with Outstanding Performance Resulting from the Synergetic Effect of Mixed P‐ and O‐Type Phases

Herein, the synthesis of new quaternary layered Na‐based oxides of the type Na_xMn_yNi_zFe_0.1Mg_0.1O₂ (0.67≤ x ≤ 1.0; 0.5≤ y ≤ 0.7; 0.1≤ z ≤ 0.3) is described. The synthesis can be tuned to obtain P2‐ and O3‐type as well as mixed P‐/O‐type phases as demonstrated by structural, morphological, and electrochemical properties characterization. Although all materials show good electrochemical performance, the simultaneous presence of the P‐ and O‐type phases is found to have a synergetic effect resulting in outstanding performance of the mixed phase material as a sodium‐ion cathode. The mixed P3/P2/O3‐type material, having an average elemental composition of Na_0.76Mn_0.5Ni_0.3Fe_0.1Mg_0.1O₂, overcomes the specific drawbacks associated with the P2‐ and O3‐type materials, allowing the outstanding electrochemical performance. In detail, the mixed phase material is able to deliver specific discharge capacities of up to 155 mAh g^?1 (18 mA g^?1) in the potential range of 2.0–4.3 V. In the narrower potential range of 2.5–4.3 V the material exhibits high average discharge potential (3.4 V versus Na/Na⁺), exceptional average coulombic efficiencies (>99.9%), and extraordinary capacity retention (90.2% after 601 cycles). The unexplored class of P‐/O‐type mixed phases introduces new perspectives for the development of layered positive electrode materials and powerful Na‐ion batteries.