N-Myc and STAT Interactor is an endometriosis suppressor.

In patients with endometriosis, refluxed endometrial fragments evade host immunosurveillance, developing into endometriotic lesions. However, the mechanisms underlying this evasion have not been fully elucidated. N-Myc and STAT Interactor (NMI) have been identified as key players in host immunosurveillance, including interferon (IFN)-induced cell death signaling pathways. NMI levels are markedly reduced in the stromal cells of human endometriotic lesions due to modulation by the Estrogen Receptor beta/Histone Deacetylase 8 axis. Knocking down NMI in immortalized human endometrial stromal cells (IHESCs) led to elevated RNA levels of genes involved in cell-to-cell adhesion and extracellular matrix signaling following IFNA treatment. Furthermore, NMI knockdown inhibited IFN-regulated canonical signaling pathways, such as apoptosis mediated by Interferon Stimulated Gene Factor 3, and necroptosis upon IFNA treatment. In contrast, NMI knockdown with IFNA treatment activated non-canonical IFN-regulated signaling pathways that promote proliferation, including ß-Catenin and AKT signaling. Moreover, NMI knockdown in IHESCs stimulated ectopic lesions' growth in mouse endometriosis models. Therefore, NMI is a novel endometriosis suppressor, enhancing apoptosis and inhibiting proliferation and cell adhesion of endometrial cells upon IFN exposure.

Flavonoids Quercetin and Kaempferol Are NR4A1 Antagonists and Suppress Endometriosis in Female Mice.

Nuclear receptor 4A1 (NR4A1) plays an important role in endometriosis progression; levels of NR4A1 in endometriotic lesions are higher than in normal endometrium, and substituted bis-indole analogs (NR4A1) antagonists suppress endometriosis progression in mice with endometriosis. In addition, the flavonoids kaempferol and quercetin are natural products that directly bind NR4A1 and significantly repress the intrinsic NR4A1-dependent transcriptional activity in human endometriotic epithelial and stromal cells and Ishikawa endometrial cancer cells. NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin suppressed proliferation of human endometriotic epithelial cells and Ishikawa cells by inhibiting epidermal growth factor receptor/c-Myc/survivin-mediated growth-promoting and survival pathways, The mammalian target of rapamycin (mTOR) signaling and αSMA/CTGF/COL1A1/FN-mediated fibrosis signaling but increasing Thioredoxin domain Containing 5/SESN2-mediated oxidative/estrogen receptors stress signaling. In human endometriotic stromal cells, NR4A1 knockdown and inhibition of NR4A1 by kaempferol and quercetin primarily inhibited mTOR signaling by suppressing proliferation of human endometrial stromal cells. In addition, kaempferol and quercetin treatment also effectively suppressed the growth of endometriotic lesions in mice with endometriosis compared with the vehicle without any body weight changes. Therefore, kaempferol and quercetin are NR4A1 antagonists with potential as nutritional therapy for endometriosis.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

Oleuropein suppresses endometriosis progression and improves the fertility of mice with endometriosis.

BACKGROUND: Endometriosis is an estrogen-dependent inflammatory reproductive disease. Therefore, systematic estrogen depletion and anti-inflammatory drugs are the current treatment for endometriosis. However, current endometriosis treatments have low efficacy and cause adverse effects in endometriosis patients. Consequently, alternative endometriosis treatments targeting endometriosis-specific factors are in demand. In this context, ERß was selected as a druggable target for endometriosis due to its critical role in progression. Therefore, selective targeting of ERß without inhibiting ERα activity would be a new paradigm for endometriosis treatment to overcome the low efficacy and adverse effects of hormonal endometriosis therapy. METHODS: Cell-based ERß and ERα activity assay systems were employed to define a selective ERß-inhibiting chemical product from a library of natural products. A surgically induced endometriosis mouse model was used to determine whether an ERß inhibitory drug suppressed endometriosis progression. Mice with endometriosis were randomly separated and then orally treated with vehicle or 25 mg/kg oleuropein (once a day for 21 days), an ERß inhibitory drug. The volume of endometriotic lesions or luciferase activity of endometriotic lesions was examined to define the growth of ectopic lesions in mice with endometriosis. The metabolite and levels of metabolic enzymes of the liver and kidney were determined in the serum of female mice treated with vehicle and oleuropein (25 mg/kg, once a day for 21 days) to define the toxicity of oleuropein. The in vitro decidualization assay was conducted with normal human endometrial stromal cells and endometriotic stromal cells to determine whether oleuropein overcomes decidualization in endometriosis patients. The pregnancy rate and pup numbers of C57BL/6 J female mice with endometriosis treated with vehicle or oleuropein (n = 10/group) were determined after mating with male mice. The cytokine profile in endometriotic lesions treated with vehicle and oleuropein (25 mg/kg) was determined with a Mouse Cytokine Array Kit. RESULTS: Among natural products, oleuropein selectively inhibited ERß but not ERα activity in vitro. Oleuropein treatment inhibited the nuclear localization of ERß in human endometrial cells upon estradiol treatment. Oleuropein (25 mg/kg) treatment suppressed the growth of mouse (6.6-fold) and human (sixfold) ectopic lesions in mice with endometriosis compared to the vehicle by inhibiting proliferation and activating apoptosis in endometriotic lesions. Oleuropein treatment did not cause reproductive toxicity in female mice. Additionally, mice with endometriosis subjected to oleuropein treatment had a higher pregnancy rate (100%) than vehicle-treated mice (70%). Furthermore, oleuropein treatment partially recovered the decidualization impact of human endometriotic stromal cells from endometriotic lesions compared to the vehicle. Oleuropein-treated mice with endometriosis exhibited significantly lower levels of cytokines directly regulated by ERß in ectopic lesions than vehicle-treated mice, illustrating the improvement in the hyperinflammatory state of mice with endometriosis. CONCLUSIONS: Oleuropein is a promising and novel nutraceutical product for nonhormonal therapy of endometriosis because it selectively inhibits ERß, but not ERα, to suppress endometriosis progression and improve the fertility of mice with endometriosis.

Interferon Signaling in the Endometrium and in Endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease that develops in reproductive-aged women who experience pelvic pain and infertility. Even though endometriosis is not a new disease, its molecular etiology has not been clearly elucidated. Defects in the immune system might be one of the factors that promote endometriosis progression. For example, elevated levels of proinflammatory cytokines are associated with endometriosis. Interferon is one of the cytokines that is elevated in endometriotic tissues compared with normal endometrium. Therefore, high interferon levels play a crucial role in endometriosis progression. In addition to endometriosis, however, interferon has a critical role in endometrial function, particularly in the initiation and maintenance of pregnancy. Therefore, this review describes the double-edged sword of interferon signaling in normal endometrial function versus endometriosis progression and also discusses interferon targeting as a new nonhormonal therapy for endometriosis. This approach may increase the efficacy of endometriosis treatment and reduce the adverse effects associated with current hormonal therapy for this disease.

Effects of Nanobubbles in Dermal Delivery of Drugs and Cosmetics.

Dermal delivery, which delivers drugs and cosmetics through the skin, has attracted significant attention due to its non-invasive and simple administration compared with oral or injectable administration. However, delivery of the ingredients through the skin barrier is difficult because the primary function of the skin is to protect the human body by preventing the invasion of contaminants. Although various techniques have been developed to overcome skin barriers, chemical toxicity, complicated processes, and expensive equipment still remain as obstacles. Moreover, green chemistry, which minimizes or eliminates the use of toxic chemicals, is required in the cosmetic industry. Thus, the development of a new method for dermal delivery is required. In this study, we provide a new method for dermal delivery using nanobubbles (NBs). NBs generated in oil improve the delivery effect of the active ingredients through the high Brownian motion and charge-balancing effect. Franz cell experiments and depigmentation experiments using the B16F10 melanoma cells were conducted to confirm the enhanced delivery effects. The system using NBs will contribute to the advancement of the dermal delivery of drugs and cosmetics.

Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration2) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.

Estrogen Inhibits Epithelial Progesterone Receptor-Dependent Progestin Therapy Efficacy in a Mouse Model of Cervical Cancer.

Although the uterine cervix responds to the female sex hormone change, the role of progesterone in cervical cancer is poorly understood. It has been shown that medroxyprogesterone acetate (MPA) regresses cervical cancer in the transgenic mouse model expressing human papillomavirus type 16 E6 and E7 oncogenes. As MPA interacts most strongly with progesterone receptor (PR), we reasoned that PR would contribute to MPA-induced regression of cervical cancer. We also hypothesized that estrogen influences the therapeutic activity of MPA because it promotes cervical cancer growth in the same mouse model. The present study showed that the deletion of Pgr in the cervical cancer cells ablated the MPA's therapeutic effect in the human papillomavirus transgenic mouse model. Additionally, estrogen attenuated cancer regression by MPA in the same model system. These observations indicate that MPA can effectively regress cervical cancer only when cancer cells express PR and estrogen levels are low. These results suggest that, if translatable, MPA should be administered when estrogen levels are low in patients with PR-positive cervical cancer.

An overview on non-spherical semiconductors for heterogeneous photocatalytic degradation of organic water contaminants.

Because of their carcinogenicity and mutagenicity, the elimination of organic contaminants from surface and subsurface water is a subject of environmental significance. Conventional water decontamination approaches such as membrane separation, ultrafiltration, adsorption, reverse osmosis, coagulation, etc., have relatively higher operating costs and can generate highly toxic secondary contaminants. On the other hand, heterogeneous photocatalysis, an advanced oxidation process (AOP), is considered a clean and cost-effective process for organic pollutants degradation. Owing to their distinctive structure and physicochemical properties non-spherical semiconductors have gained considerable limelight in the photocatalytic degradation of organic contaminants. The current review briefly introduces a wide range of organic water contaminants. Recent advances in non-spherical semiconductor assembly and their photocatalytic degradation applications are highlighted. The underlying mechanism, fundamentals of photocatalytic reactions, and the factors affecting the degradation performance are also alluded including the current challenges and future research perspectives.

Quantification and Metabolite Identification of Sulfasalazine in Mouse Brain and Plasma Using Quadrupole-Time-of-Flight Mass Spectrometry.

Sulfasalazine (SAS), an anti-inflammatory drug with potent cysteine/glutamate antiporter system xc-(SXC) inhibition has recently shown beneficial effects in brain-related diseases. Despite many reports related to central nervous system (CNS) effect of SAS, pharmacokinetics (PK) and metabolite identification studies in the brain for SAS were quite limited. The aim of this study was to investigate the pharmacokinetics and metabolite identification of SAS and their distributions in mouse brain. Using in vivo brain exposure studies (neuro PK), the PK parameters of SAS was calculated for plasma as well as brain following intravenous and oral administration at 10 mg/kg and 50 mg/kg in mouse, respectively. In addition, in vivo metabolite identification (MetID) studies of SAS in plasma and brain were also conducted. The concentration of SAS in brain was much lower than that in plasma and only 1.26% of SAS was detected in mouse brain when compared to the SAS concentration in plasma (brain to plasma ratio (%): 1.26). In the MetID study, sulfapyridine (SP), hydroxy-sulfapyridine (SP-OH), and N-acetyl sulfapyridine (Ac-SP) were identified in plasma, whereas only SP and Ac-SP were identified as significant metabolites in brain. As a conclusion, our results suggest that the metabolites of SAS such as SP and Ac-SP might be responsible for the pharmacological effect in brain, not the SAS itself.

Progesterone Receptor Is a Haploinsufficient Tumor-Suppressor Gene in Cervical Cancer.

Tumor-suppressor genes (TSG) are often deleted or transcriptionally suppressed in cancer. PGR codes for progesterone receptor (PR), a transcription factor whose function depends on its ligand. Although PR expression is often undetectable in cervical cancer, its relevance to the endocrine-related etiology of this prevalent gynecological disease remains unclear. In this study, we show that the deletion of one Pgr allele in cervical epithelium promoted spontaneous cervical cancer in human papilloma viral oncogene-expressing transgenic mice as efficiently as the ablation of both Pgr alleles. We also show that tumors arising in the transgenic mice with one or both Pgr alleles did not express PR or expressed at the reduced levels compared with the normal epithelium. PR status correlated with estrogen receptor α (ERα) status in the mouse model and the Cancer Genome Atlas (TCGA) dataset. TCGA data analyses revealed that PGR expression significantly decreased in cervical cancer and that the biallelic deletion of PGR was rare. Furthermore, low PGR expression was associated with poor prognosis in young patients with cervical cancer. These discoveries point to PGR as a haploinsufficient TSG in the uterine cervix. They also raise the possibility that the restoration of PGR expression may improve the survival rate. IMPLICATIONS: The decreased expression of PR may increase the risk of cervical cancer in human papillomavirus-infected women. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/42/F1.large.jpg.

N-nitrosoethylenethiourea formation at environmentally-relevant concentrations of ethylenethiourea in a pooled groundwater sample.

N-nitroso compounds form from the interaction between nitrosatable precursors and nitrite under acidic conditions. A majority of N-nitroso compounds tested show evidence of carcinogenicity in animal models. Formation of N-nitroso compounds may occur from exposure to precursors in drinking water, but the extent of formation depends on a number of factors, including concentration of substrates, presence of catalysts and inhibitors, and pH. The objective of this study was to examine these factors in pesticide-associated N-nitroso (PANN) compound formation in drinking water. In preliminary screening experiments, nine nitrosatable pesticides and degradation products were individually reacted at environmentally-relevant concentrations (≤ 20 µg L--1) with sodium nitrite (NaNO2) and hydrochloric acid (HCl) in ultra-pure water. Only ethylenethiourea (ETU) showed evidence of PANN compound formation in initial experiments and was further tested for N-nitrosoethylenethiourea (N-ETU) formation in a pooled groundwater sample (comprised of five tap water samples combined into one homogenous sample) collected from an agricultural region of Prince Edward Island in Canada, where nitrate contamination is a known concern. Evidence of N-ETU formation in the groundwater sample was observed within 30 min at concentrations 7.5, 10, and 20 µg L-1. Analysis of target compounds and semi-target PANN compounds was performed using ultra-high pressure liquid chromatography coupled with high resolution orbital ion trap mass spectrometry. These preliminary experiments serve to inform about potential PANN compound formation in groundwater. The results of this study suggest that ETU is capable of forming potentially carcinogenic N-ETU in water containing nitrite/nitrate at trace concentrations under acidic conditions. Thus, these findings suggest that N-ETU formation may be a concern for individuals exposed to low concentrations of ETU in groundwater.

Assessment of Pharmacokinetics and Metabolism Profiles of SCH 58261 in Rats Using Liquid Chromatography-Mass Spectrometric Method.

5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is one of the new chemical entities that has been developed as an adenosine A2A receptor antagonist. Although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 from a drug metabolism or pharmacokinetics perspective. This study describes the metabolism and pharmacokinetic properties of SCH 58261 in order to understand its behaviors in vivo. Rats were used as the in vivo model species. First, an LC-MS/MS method was developed for the determination of SCH 58261 in rat plasma. A GastroPlus™ simulation, in vitro microsomal metabolic stability, and bile duct-cannulated studies were also performed to understand its pharmacokinetic profile. The parameter sensitivity analysis of GastroPlus™ was used to examine the factors that influence exposure when the drug is orally administered. The factors are as follows: permeability, systemic clearance, renal clearance, and liver first-pass effect. In vitro microsomal metabolic stability indicates how much the drug is metabolized. The extrapolated hepatic clearance value of SCH 58261 was 39.97 mL/min/kg, indicating that the drug is greatly affected by hepatic metabolism. In vitro microsomal metabolite identification studies revealed that metabolites produce oxidized and ketone-formed metabolites via metabolic enzymes in the liver. The bile duct-cannulated rat study, after oral administration of SCH 58261, showed that a significant amount of the drug was excreted in feces. These results imply that the drug is not absorbed well in the body after oral administration. Taken together, SCH 58261 showed quite a low bioavailability when administered orally and this was likely due to significantly limited absorption, as well as high metabolism in vivo.

Liquid chromatography-high resolution mass spectrometric method for the quantification of monomethyl auristatin E (MMAE) and its preclinical pharmacokinetics.

MMAE is a potent antimitotic drug used as payload of an antibody-drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography-high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography - quadrupole - time-of-flight - tandem mass spectrometry (LC-qTOF-MS/MS) analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2 ), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1.01-2,200 ng/mL for MMAE. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. Recovery was 42.84%. The dilution integrity was determined for 5-fold dilution and the accuracy and precision ranged within ±25%. The stability results indicated that MMAE was stable for the following conditions: short-term (4 h), long-term (4 weeks), freeze/thaw (3 cycles) and post-preparative stability (12 h). This qualified method was successfully applied to a pharmacokinetic study of MMAE in rat as a preclinical animal model. The PK results suggest that MMAE has moderate CL and low BA.Also, these results would be helpful in having a comprehensive understanding of the PK characteristics of MMAE and developing ADC in future.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015-2018.

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015-2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL-1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 102.5 EID50 /0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South-East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.

Comparison of sample preparation approaches and validation of an extraction method for nitrosatable pesticides and metabolites in human serum and urine analyzed by liquid chromatography - Orbital ion trap mass spectrometry.

In the acidic environment of the stomach, nitrosatable pesticide residues may react with nitrite to form potentially carcinogenic pesticide-associated N-nitroso compounds (PANOCs). The objective of this study was to develop a method for the analysis of 10 nitrosatable pesticides and breakdown products in human serum and urine. Three sample preparation methods were evaluated for extraction of target analytes from the biomatrices. Deproteinization by methanol for 300-µL aliquots of serum with a final extract volume of 225 µL resulted in excessive ion enhancement of some analytes and suppression of others. Three types of solid-phase extraction cartridges were tested for optimal analyte retention from 200-µL aliquots of serum with a final extract volume of 400 µL; this approach resulted in significant analyte loss for some compounds. The Quick, Easy, Cheap, Effective, Rugged, and Safe approach resulted in a suitable method for extraction of the analytes from each biomatrix. Biofluid samples (500 µL) were spiked to 100 µg L-1 with analytical standards and extracted using 500 µL of acetonitrile (ACN) with 4% acetic acid (AcOH) for serum and 0.1% AcOH in ACN for urine. For extraction, 200 mg magnesium sulfate (MgSO4) and 50 mg sodium acetate were added for serum and 200 mg MgSO4 and 50 mg sodium chloride were added for urine. Final extract volumes for both biomatrices using the QuEChERS method was 400 µL after dilution. Samples were analyzed via ultra-high pressure liquid chromatography/high-resolution accurate mass orbital ion trap mass spectrometry. Mean recoveries for target analytes in serum and urine ranged between 74 and 120% (%RSD < 12) and 96 to 116% (%RSD ≤ 10), respectively. These methods may be used in large-scale biomonitoring studies to analyze PANNs and their parent compounds in human serum and urine.

Quantitative Analysis of Tozadenant Using Liquid Chromatography-Mass Spectrometric Method in Rat Plasma and Its Human Pharmacokinetics Prediction Using Physiologically Based Pharmacokinetic Modeling.

Tozadenant is one of the selective adenosine A2a receptor antagonists with a potential to be a new Parkinson's disease (PD) therapeutic drug. In this study, a liquid chromatography-mass spectrometry based bioanalytical method was qualified and applied for the quantitative analysis of tozadenant in rat plasma. A good calibration curve was observed in the range from 1.01 to 2200 ng/mL for tozadenant using a quadratic regression. In vitro and preclinical in vivo pharmacokinetic (PK) properties of tozadenant were studied through the developed bioanalytical methods, and human PK profiles were predicted using physiologically based pharmacokinetic (PBPK) modeling based on these values. The PBPK model was initially optimized using in vitro and in vivo PK data obtained by intravenous administration at a dose of 1 mg/kg in rats. Other in vivo PK data in rats were used to validate the PBPK model. The human PK of tozadenant after oral administration at a dose of 240 mg was simulated by using an optimized and validated PBPK model. The predicted human PK parameters and profiles were similar to the observed clinical data. As a result, optimized PBPK model could reasonably predict the PK in human.

Fluorescence Spectra Predict Microcystin-LR and Disinfection Byproduct Formation Potential in Lake Water.

Disinfection byproducts (DBPs) and algal toxins can be expensive to monitor and represent significant potential risks to human health. DBPs, including haloacetic acids and trihalomethanes, are possible or probable human carcinogens. Microcystin-LR-produced by cyanobacteria-is linked with various adverse health effects. Here we show that fluorescence spectra predict both microcystin-LR occurrence and DBP formation potential (DBPfp) in lake water. We compared models with either fluorescence spectra or a suite of water quality predictors as inputs. A regularized logistic regression model with fluorescence spectral inputs correctly classified 94% of test data with respect to microcystin-LR occurrence, with a 96% probability of correctly ranking a detect/nondetect pair. Regularized linear regression predicted DBPfp based on fluorescence inputs with a combined R2 of 0.83 on test data. A gradient-boosted classifier with seven water quality inputs was comparable in detecting microcystin-LR (91% correct), as was UV254 in predicting DBPfp (combined test R2 = 0.84), but no single parameter matched fluorescence spectra over both predictive tasks. Results highlight the potential for multiparameter monitoring via fluorescence spectroscopy, extending previous work on predicting DBPs alone. As a high-frequency monitoring tool, this approach could supplement mass spectrometric methods that may only be applicable at low frequency due to resource limitations.

Epithelial oestrogen receptor α is dispensable for the development of oestrogen-induced cervical neoplastic diseases.

Human papillomavirus (HPV) is required but not sufficient for cervical carcinoma (CxCa) development. Oestradiol (E2 ) promotes CxCa development in K14E7 transgenic mice expressing the HPV16 E7 oncoprotein under the control of the keratin (K14) promoter. E2 mainly functions through oestrogen receptor α (ERα). However, the role of ERα in human CxCa has been underappreciated largely because it is not expressed in carcinoma cells. We have shown that deletion of Esr1 (the ERα-coding gene) in the cervical stroma of K14E7 mice promotes regression of cervical intraepithelial neoplasia (CIN), the precursor lesion of CxCa. Here, we deleted Esr1 in the cervical epithelium but not in the stroma. We found that E2 induced cervical epithelial cell proliferation in epithelial ERα-deficient mice. We also found that E2 promoted the development of CIN and CxCa in epithelial ERα-deficient K14E7 mice and that all neoplastic epithelial cells were negative for ERα. In addition, proliferation indices were similar between ERα- and ERα+ CxCa. These results indicate that epithelial ERα is not necessary for E2 -induced CIN and CxCa. Taking these findings together, we conclude that stromal ERα rather than epithelial ERα mediates oncogenic E2 signalling in CxCa. Our results support stromal ERα signalling as a therapeutic target for the disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.