Kinetic analysis of mass transfer of Eu(III) in extractant-impregnated polymer-layered silica particle in multiple-ion distribution system.

The Eu(III) distribution mechanism in single extractant-impregnated polymer-layered silica particle in a complex solution containing multiple lanthanide ions was investigated using fluorescence microspectroscopy, which was compared with the single-ion distribution system. The rate-determining step of the Eu(III) distribution was the reaction of Eu(III) with the two extractant molecules in the particle. The distribution mechanism and rate constants obtained in the multiple lanthanide ions-distribution system agreed with those of the single-ion distribution system.

Eu(III) transfer in single N,N,N',N'-tetraoctyldiglycolamide-impregnated polymer-coated silica particle using fluorescence microspectroscopy: transfer mechanism and effect of polymer crosslinking degree.

A microcapillary manipulation system combined with fluorescence microspectroscopy enabled us to analyze mass transfer in a single particle. In this study, we revealed the Eu(III) distribution in a single diglycolamide-derivative extractant (TODGA)-impregnated polymer-coated silica particle. The reaction of Eu(III) with two TODGA molecules in the polymer layer was the rate-limiting process, which was revealed by the relationship between the rate constants (k1 and k-1) and concentrations of Eu(III) and HNO3. The decrease in the crosslinking degree of the polymer layer caused an increase in only k-1. This indicates that hydrophilic environments at lower crosslinking degrees enhance the stability of the charged Eu(III) species such as Eu3+, Eu(NO3)2+, and Eu(NO3)2+.

Improvement in the elution performance of an N,N,N',N-tetraoctyl diglycolamide impregnated extraction chromatography adsorbent using neodymium via micro-particle-induced X-ray emission analysis.

An adsorbent used for the recovery of trivalent minor actinides (MA(III); Am and Cm) from high level liquid waste generated from reprocessing of spent nuclear fuel was subjected to micro-particle induced X-ray emission (micro-PIXE) analysis to improve its elution performance. The experimental adsorbent comprised SiO2 particles, a polymer coating, and an N,N,N',N'-tetraoctyl diglycolamide (TODGA). The particles to be analyzed were subjected to Nd adsorption and an elution operation, but Nd in the adsorbent was found to be uniformly distributed. This might have been caused by individual differences in the amount of impregnated TODGA. The remaining Nd species were not localized to a specific part of the adsorbent after the adsorption operation. Some Nd elements were retained in the adsorbent after elution, probably because of the poor diffusion of the mobile phase inside the adsorbent. An adsorbent having a different microstructure from the first was then evaluated, and rapid elution was observed on new adsorbent along micro-PIXE analysis.

Prompt Reduction in CRP, IL-6, IFN-γ, IP-10, and MCP-1 and a Relatively Low Basal Ratio of Ferritin/CRP Is Possibly Associated With the Efficacy of Tocilizumab Monotherapy in Severely to Critically Ill Patients With COVID-19.

Background and Aim: Tocilizumab, a humanized anti-IL-6 receptor antibody, has been used to treat severely to critically ill patients with COVID-19. A living systematic review with meta-analysis of recent RCTs indicates that the combination therapy of corticosteroids and tocilizumab produce better outcomes, while previous observational studies suggest that tocilizumab monotherapy is beneficial for substantial numbers of patients. However, what patients could respond to tocilizumab monotherapy remained unknown. Methods: In this retrospective study we evaluated the effects of tocilizumab monotherapy on the clinical characteristics, serum biomediator levels, viral elimination, and specific IgG antibody induction in 13 severely to critically ill patients and compared with those of dexamethasone monotherapy and dexamethasone plus tocilizumab. Results: A single tocilizumab administration led to a rapid improvement in clinical characteristics, inflammatory findings, and oxygen supply in 7 of 11 patients with severe COVID-19, and could recover from mechanical ventilation management (MVM) in 2 patients with critically ill COVID-19. Four patients exhibited rapidly worsening even after tocilizumab administration and required MVM and additional methylprednisolone treatment. Tocilizumab did not delay viral elimination or inhibit IgG production specific for the virus, whereas dexamethasone inhibited IgG induction. A multiplex cytokine array system revealed a significant increase in the serum expression of 54 out of 80 biomediators in patients with COVID-19 compared with that in healthy controls. Compared with those who promptly recovered in response to tocilizumab, patients requiring MVM showed a significantly higher ratio of basal level of ferritin/CRP and a persistent increase in the levels of CRP and specific cytokines and chemokines including IL-6, IFN-γ, IP-10, and MCP-1. The basal high ratio of ferritin/CRP was also associated with clinical deterioration even in patients treated with dexamethasone and tocilizumab. Conclusion: Tocilizumab as monotherapy has substantial beneficial effects in some patients with severe COVID-19, who showed a relatively low level of the ratio of ferritin/CRP and prompt reduction in CRP, IL-6, IFN-γ, IP-10, and MCP-1. The high ratio of ferritin/CRP is associated with rapid worsening of pneumonia. Further evaluation is warranted to clarify whether tocilizumab monotherapy or its combination with corticosteroid is preferred for severely to critically ill patients with COVID-19.

Complexation-Distribution Separated Solvent Extraction Process Designed for Rapid and Efficient Recovery of Inert Platinum Group Metals.

Previously, we have demonstrated that thermal-assisted techniques can accelerate the extraction of inert platinum group metals (PGMs), while they still have several concerns about difficulty of temperature control in actual extraction contactors and safety risks arising from heating organic solvents. In this study, we report a complexation-distribution separated extraction process for the accelerated extraction of inert PGMs. This extraction method includes two steps: (1) complexation of PGMs with extractants in aqueous solution and (2) distribution of the formed complex from the aqueous phase to organic one. We separately investigated the complexation and distribution processes for typical inert PGMs such as Ru(III) and Rh(III) in the presence of water-soluble N,N,N',N'-tetra-alkylpyridinediamide ligands (PDA) and bis(trifluoromethylsulfonyl)amide (Tf2N-) counteranions. As a result, the water-soluble complexes of Ru(III) and Rh(III) with PDA can be formed in 0.5 M HNO3(aq) within 3 h under heating at 356 K. The formed complexes were extracted to the 1-octanol layer containing Tf2N- within 5 min at room temperature, where this hydrophobic anion plays an important role to promote extraction of PGMs as an anionic phase-transfer catalyst (PTC). Consequently, we successfully established and demonstrated the complexation-distribution separated extraction process for the accelerated extraction of inert PGMs using a water-soluble ligand and anionic PTC.

Scoring system for identifying Japanese patients with COVID-19 at risk of requiring oxygen supply: A retrospective single-center study.

INTRODUCTION: Japan is facing the threat of medical system collapse due to the rapid spread of coronavirus disease 2019 (COVID-19). The present scoring system may help assess disease severity and oxygen supply requirements in COVID-19 patients. METHODS: Data on patient characteristics at baseline and throughout hospitalization for COVID-19 were extracted from medical records. Disease severity was dichotomized into two categories without or with oxygen supply as asymptomatic, mild, and moderate illness (AMMI), and severe and critical illness (CSI). The AMMI and CSI groups were compared. Predictors of disease severity, previously identified in the outpatient setting, were included in multivariable logistic regression analysis; the obtained coefficients were converted to integers and assigned a score. RESULTS: A total of 206 patients diagnosed with COVID-19 were included in this study. Correlation between COVID-19 severity and medical information was examined by comparing AMMI and CSI. Age, hemodialysis, and C-reactive protein (CRP) levels were candidate predictors of the need for oxygen supply in patients with COVID-19. Coefficients associated with age, hemodialysis, and CRP were as follows: 1 × age (in years, coded as 0 for values of <50, and as 1 for values of ≥50) + 1 × hemodialysis (coded as 0 for "no", and as 1 for "yes") + 1 × CRP (in mg/dL, coded as 0 for values of <1.0, and as 1 for values of ≥1.0). Patients with scores of ≥2 points required oxygen supply (sensitivity, 68.4%; specificity, 79.0%) CONCLUSION: The present model can help predict disease severity and oxygen requirements in COVID-19 patients in Japan.

Clinical COVID-19 diagnostic methods: Comparison of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and quantitative RT-PCR (qRT-PCR).

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is a major public health concern. Accurate and rapid diagnosis of COVID-19 is critical for disease control. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification assay similar to reverse transcription-polymerase chain reaction (RT-PCR), the former being a simple, low cost, and rapid method. OBJECTIVES: This study aimed to compare the RT-LAMP assay with RT-PCR using the Loopamp™ SARS-CoV-2 Detection Kit. STUDY DESIGN: One hundred and fifty-one nasopharyngeal swab and 88 sputum samples obtained from individuals with suspected or confirmed COVID-19 were examined. RESULTS: RT-LAMP had high specificity (98.5 % (95 % CI: 96.9-100 %)), sensitivity (87.0 % (95 % CI: 82.8-91.3 %)), positive predictive value (97.9 % (95 % CI: 96.1-99.7 %)), negative predictive value (90.2 % (95 % CI: 86.4-94.0 %)), and concordance rate (93.3 % (95 % CI: 90.1-96.5 %)). Nasopharyngeal and sputum samples positive in RT-LAMP contained as few as 10.2 and 23.4 copies per 10 µL, respectively. RT-LAMP showed similar performance to RT-PCR for samples with cycle threshold value below 36. CONCLUSIONS: These results indicate that RT-LAMP is a highly reliable and at least equivalent to RT-PCR in utility, and potentially applicable in settings that are more diverse as a point-of-care tool.

Factors significantly associated with COVID-19 severity in symptomatic patients: A retrospective single-center study.

INTRODUCTION: The severity of coronavirus disease (COVID-19) in Japanese patients is unreported. We retrospectively examined significant factors associated with disease severity in symptomatic COVID-19 patients (COVID-Pts) admitted to our institution between February 20 and April 30, 2020. METHODS: All patients were diagnosed based on the genetic detection of severe acute respiratory syndrome coronavirus 2. Information on the initial symptoms, laboratory data, and computed tomography (CT) images at hospitalization were collected from the patients' records. COVID-Pts were categorized as those with critical or severe illness (Pts-CSI) or those with moderate or mild illness (Pt-MMI). All statistical analyses were performed using R software. RESULTS: Data from 61 patients (16 Pt-CSI, 45 Pt-MMI), including 58 Japanese and three East Asians, were analyzed. Pt-CSI were significantly older and had hypertension or diabetes than Pt-MMI (P < 0.001, 0.014 and < 0.001, respectively). Serum albumin levels were significantly lower in Pt-CSI than in Pt-MMI (P < 0.001), whereas the neutrophil-to-lymphocyte ratio and C-reactive protein level were significantly higher in Pt-CSI than in Pt-MMI (P < 0.001 and P < 0.001, respectively). In the CT images of 60 patients, bilateral lung lesions were more frequently observed in Pt-CSI than in Pt-MMI (P = 0.013). Among the 16 Pt-CSI, 15 received antiviral therapy, 12 received tocilizumab, five underwent methylprednisolone treatment, six received mechanical ventilation, and one died. CONCLUSIONS: The illness severity of Japanese COVID-Pts was associated with older age, hypertension and/or diabetes, low serum albumin, high neutrophil-to-lymphocyte ratio, and C-reactive protein.

Extraction Mechanism of Lanthanide Ions into Silica-based Microparticles Studied by Single Microparticle Manipulation and Microspectroscopy.

Single porous silica microparticles coated with a styrene-divinylbenzene polymer (SDB) impregnated with octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) were injected into an aqueous 3 mol/L nitric acid solution containing trivalent lanthanide (Ln(III)), as a high-level liquid waste model. We used the microcapillary manipulation-injection technique; and the extraction rate of Ln(III), as an Ln(III)-CMPO complex, into the single microparticles was measured by luminescence microspectroscopy. The extraction rate significantly depended on the Ln(III), CMPO, or NO3- concentration, and was analyzed in terms of diffusion in the pores of the microparticles and the complex formation of Ln(III). The results indicated that the rate-determining step in Ln(III) extraction was diffusion in the pore solution of the microparticles.

Extraction of Pd(II), Rh(III) and Ru(III) from HNO(3) aqueous solution to betainium bis(trifluoromethanesulfonyl)imide ionic liquid.

Extraction efficiencies of Pd(ii), Rh(iii), and Ru(iii) from HNO3(aq) to [Hbet][Tf2N] were demonstrated, i.e., Pd(ii) is the most extractable, Rh(iii) is medium extractable, and Ru(iii) is the least extractable. The extraction seems to proceed through coordination of betaine to the metal ions and the cation exchange of the formed complex with H(+).

Spectroelectrochemical identification of a pentavalent uranyl tetrachloro complex in room-temperature ionic liquid.

Reduction of U(VI)O(2)Cl(4)(2-) in a mixture of 1-ethyl-3-methylimidazolium tetrafluoroborate and its chloride at E°' = -0.996 V vs Fc/Fc(+) and 298 K affords U(V)O(2)Cl(4)(3-), which is kinetically stable and exhibits typical character of U(V) in the UV-vis-NIR absorption spectrum.

Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo.

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo.

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil.

MK-1775 is a potent and selective small molecule Wee1 inhibitor. Previously we have shown that it abrogated DNA damaged checkpoints induced by gemcitabine, carboplatin, and cisplatin and enhanced the anti-tumor efficacy of these agents selectively in p53-deficient tumor cells. MK-1775 is currently in Phase I clinical trial in combination with these anti-cancer drugs. In this study, the effects of MK-1775 on 5-fluorouracil (5-FU) and other DNA-damaging agents with different modes of action were determined. MK-1775 enhanced the cytotoxic effects of 5-FU in p53-deficient human colon cancer cells. MK-1775 inhibited CDC2 Y15 phosphorylation in cells, abrogated DNA damaged checkpoints induced by 5-FU treatment, and caused premature entry of mitosis determined by induction of Histone H3 phosphorylation. Enhancement by MK-1775 was specific for p53-deficient cells since this compound did not sensitize p53-wild type human colon cancer cells to 5-FU in vitro. In vivo, MK-1775 potentiated the anti-tumor efficacy of 5-FU or its prodrug, capecitabine, at tolerable doses. These enhancements were well correlated with inhibition of CDC2 phosphorylation and induction of Histone H3 phosphorylation in tumors. In addition, MK-1775 also potentiated the cytotoxic effects of pemetrexed, doxorubicin, camptothecin, and mitomycin C in vitro. These studies support the rationale for testing the combination of MK-1775 with various DNA-damaging agents in cancer patients.

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Discovery of gene expression-based pharmacodynamic biomarker for a p53 context-specific anti-tumor drug Wee1 inhibitor.

BACKGROUND: Wee1 is a tyrosine kinase regulating S-G2 cell cycle transition through the inactivating phosphorylation of CDC2. The inhibition of Wee1 kinase by a selective small molecule inhibitor significantly enhances the anti-tumor efficacy of DNA damaging agents, specifically in p53 negative tumors by abrogating S-G2 checkpoints, while normal cells with wild-type p53 are not severely damaged due to the intact function of the G1 checkpoint mediated by p53. Since the measurement of mRNA expression requires a very small amount of biopsy tissue and is highly quantitative, the development of a pharmacodynamic (PD) biomarker leveraging mRNA expression is eagerly anticipated in order to estimate target engagement of anti-cancer agents. RESULTS: In order to find the Wee1 inhibition signature, mRNA expression profiling was first performed in both p53 positive and negative cancer cell lines treated with gemcitabine and a Wee1 inhibitor, MK-1775. We next carried out mRNA expression profiling of skin samples derived from xenograft models treated with the Wee1 inhibitor to identify a Wee1 inhibitor-regulatory gene set. Then, the genes that were commonly modulated in both cancer cell lines and rat skin samples were extracted as a Wee1 inhibition signature that could potentially be used as a PD biomarker independent of p53 status. The expression of the Wee1 inhibition signature was found to be regulated in a dose-dependent manner by the Wee1 inhibitor, and was significantly correlated with the inhibition level of a direct substrate, phosphorylated-CDC2. Individual genes in this Wee1 inhibition signature are known to regulate S-G2 cell cycle progression or checkpoints, which is consistent with the mode-of-action of the Wee1 inhibitor. CONCLUSION: We report here the identification of an mRNA gene signature that was specifically changed by gemcitabine and Wee1 inhibitor combination treatment by molecular profiling. Given the common regulation of expression in both xenograft tumors and animal skin samples, the data suggest that the Wee1 inhibition gene signature might be utilized as a quantitative PD biomarker in both tumors and surrogate tissues, such as skin and hair follicles, in human clinical trials.

Identification of beta-catenin as a novel substrate of Polo-like kinase 1.

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays an important role in M phase progression by regulating various downstream substrates via phosphorylation. Here, we identified beta-catenin as a novel substrate of Plk1 and determined that Ser-718 is a phosphorylation site for Plk1 by using a phospho-specific antibody that cross-reacts with Plk1-dependent phosphorylation sites. Ser-718 of beta-catenin was directly phosphorylated by recombinant Plk1 in vitro, with the phosphorylation signal in cells increasing with overexpression of Plk1 and decreasing when endogenous Plk1 was depleted by small interfering RNA. The phosphorylation at Ser-718 was correlated with the cell cycle-dependent expression of Plk1 which reached a maximum in M phase. We also confirmed that there is a physical interaction between beta-catenin and Plk1 using coimmunoprecipitation and a GST pull-down assay. These results demonstrate that beta-catenin is a physiological substrate of Plk1 in cells, which may provide a novel insight into the role of beta-catenin in M phase.

Carcinogenicity of dimethylarsinic acid in Ogg1-deficient mice.

Oxidative stress to DNA is recognized as a mechanism underlying carcinogenic effects of some environmental agents. Here, we hypothesized that dimethylarsinic acid (DMA(V)), an organic metabolite of inorganic arsenic in humans, might exert carcinogenic potential in a mouse line carrying a mutant Mmh allele of the Mmh/OGG1 gene encoding the enzyme 8-hydroxyguanine DNA glycosylase 1 (OGG1). Ogg1 mutant and wild type mice were treated with DMA(V) in their drinking water at a dose of 200 p.p.m. for up to 72 weeks. All DMA(V)-treated Ogg1(-/-)animals developed tumors, with a tendency for lower total incidences in the Ogg1(+/+) cases. Lung tumors in particular were induced as compared to the lack in non-carcinogen controls and were significantly more frequent in the homozygotes. At week 4, the levels of DNA 8-OH-dG and cell proliferation were significantly elevated in the lungs of non-treated Ogg1(-/-) as compared to Ogg1(+/+) mice and were strongly enhanced by DMA(V) treatment. Marked induction of Pola1, Cyp7b1, Ndfua3, Mmp13 and other genes specific to cell proliferation, cell signaling and xenobiotic metabolism in the lungs of DMA(V)-treated Ogg1(-/-) mice was found. Electron microscopic examination revealed the growth of microvilli, with increased numbers of mitochondria only in lungs and lung tumors of DMA(V)-exposed Ogg1(-/-) mice. Therefore, we strongly suggest that DMA(V) exerts carcinogenicity in the lungs of Ogg1(-/-) mutant mice, with a possible role for persistent accumulation of DNA oxidative adducts.

The study using wild-type and Ogg1 knockout mice exposed to potassium bromate shows no tumor induction despite an extensive accumulation of 8-hydroxyguanine in kidney DNA.

In order to assess the effect of potassium bromate (KBrO3) on the induction of tumor formation, a 1-year carcinogenesis study was performed using Ogg1 knockout mice (Ogg1(-/-)) and wild-type mice (Ogg1(+/+)). The mice were chronically exposed to KBrO3 by putting it in the drinking water for 29 weeks, at 2 g/l for the first 18 weeks, and then at 1 g/l for another 11 weeks. After termination of treatment the mice were kept for an additional 23 weeks. The amount of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA after 29 weeks of KBrO3 exposure reached 500 8-OH-dG/10(6) dG, almost 250-fold that of untreated wild-type mice. During the course of study the mice appeared normal, although a decrease of body weight gain in both Ogg1(-/-) and Ogg1(+/+) mice exposed to KBrO3, and some kidney malfunction in KBrO3 treated Ogg1(-/-) mice was observed. Surprisingly, when Ogg1(-/-) and Ogg1(+/+) mice were sacrificed at 52 weeks, no tumor formation could be found in kidney or other organs such as lung, liver, spleen, thymus, stomach and intestine. Microscopic examination also showed the absence of precancerous foci in all tissues of both Ogg1(-/-) and Ogg1(+/+) mice. A possible explanation is presented to reconcile these results with those of others which showed an increased incidence of tumor formation in untreated Ogg1(-/-) mice.

A high-throughput phage display screening method using a combination of real-time PCR and affinity chromatography.

Phage display is one of the best methods to identify drug targets, although technical problems including imprecision in quantifying phage and false-positive results are common. To address these difficulties, we propose two methods to more rapidly identify drug-binding phage particles. First, quantification of phage using SYBR Green Real-time PCR significantly improved accuracy and reproducibility. Second, affinity-column chromatography for selection of drug-binding phage particles concentrated particles more than a 96-well plate, making a phage amplification step, which can bias phage distribution, unnecessary. The methods proposed here should be suitable for high-throughput phage-display screenings and ultimately lead to more rapid identification of drug targets.