Unconjugated Form Research Articles

Occupational exposure of cashiers to bisphenol S via thermal paper

PurposeIn thermal paper, Bisphenol S (BPS) is one of the alternatives for bisphenol A (BPA). Due to its structural similarity to BPA, concern has been raised about the safety of BPS. Indeed, handling thermal paper receipts could be a source of occupational exposure to BPS among cashiers, as it was previously described for BPA. In this study, we investigated whether frequent contacts with thermal paper are associated with an increase in urinary BPS levels in cashiers. MethodTotal (unconjugated and conjugated forms) and free (unconjugated) BPS were measured in urine samples from 17 cashiers and 15 controls, using LC–MS/MS. Spot urine samples, including pre-shift and post-sift samples and first morning void were collected from each volunteer. BPS concentration in thermal paper was determined and the number of receipts handled by cashiers was estimated as well. ResultsThe median urinary total BPS concentration was 0.67 μg/L (0.52 μg/g creatinine) for controls and 2.53 μg/L (2.07 μg/g creatinine) for cashiers. Total BPS concentration was significantly higher in cashiers than in controls. Free BPS was detected in less than 20% of urine samples collected from controls and in less than 50% of urine samples collected from cashiers. ConclusionThe detectable levels of BPS in urine of controls suggest an exposure to BPS of the general population. In addition, frequent contact with thermal paper could be responsible for an increase in urinary concentration of total BPS in cashiers.

Relationship between free and total malondialdehyde, a well-established marker of oxidative stress, in various types of human biospecimens.

Oxidative stress is involved in thoracic diseases and health responses to air pollution. Malondialdehyde (MDA) is a well-established marker of oxidative stress, but it may be present in unconjugated and conjugated forms. To our knowledge, no studies have conducted a systemic evaluation of both free MDA (unconjugated MDA) and total MDA (the sum of both unconjugated and conjugated MDA) across various types of human biospecimens. Free MDA and total MDA were simultaneously measured in a range of human biospecimens, including nasal fluid (N=158), saliva (N=158), exhaled breath condensate (N=40), serum (N=232), and urine (N=429). All samples were analyzed using an HPLC-fluorescence method with high sensitivity and specificity. Due to the right skewed distribution of free MDA and total MDA, we performed natural-log transformation before subsequent statistical analyses. The relationship between the natural log of free and total MDA was evaluated by R2 of simple linear regression. T test was used for comparisons of means between two groups. One-way analysis of variance was used in combination with Tukey's test to compare the natural log of the ratio of free MDA to total MDA across various types of biospecimens. For exhaled breath condensate, serum, urine, nasal fluid and saliva samples, the R2 between free and total MDA were 0.61, 0.22, 0.59, 0.47 and 0.06, respectively; the medians of the free MDA to total MDA ratio were 48.1%, 17.4%, 9.8%, 5.1% and 3.0%, respectively; the free MDA to total MDA ratio in EBC > serum > urine > nasal fluid > saliva (P<0.001 for pairwise comparisons). For exhaled breath condensate and urine samples, using either free or total MDA can provide information regarding the level of oxidative stress; however, that is not the case for serum, nasal fluid, and saliva given the low correlations between free and total MDA.

Development of an on-line solid phase extraction ultra-high-performance liquid chromatography technique coupled to tandem mass spectrometry for quantification of bisphenol S and bisphenol S glucuronide: Applicability to toxicokinetic investigations

Regulatory measures and public concerns regarding bisphenol A (BPA) have led to its replacement by structural analogues, such as Bisphenol S (BPS), in consumer products. At present, no toxicokinetic investigations have been conducted to assess the factors determining human internal exposure to BPS for subsequent risk assessment. Toxicokinetic studies require reliable analytical methods to measure the plasma concentrations of BPS and its main conjugated metabolite, BPS-glucuronide (BPS-G).An efficient on-line SPE-UPLC–MS/MS method for the simultaneous quantification of BPS and BPS-G in ovine plasma was therefore developed and validated in accordance with the European Medicines Agency guidelines for bioanalytical method validation. This method has a limit of quantification of 3ngmL−1 for BPS and 10ngmL−1 for BPS-G, an analytical capacity of 200 samples per day, and is particularly well suited to toxicokinetic studies.Use of this method in toxicokinetic studies in sheep showed that BPS, like BPA, is efficiently metabolized into its glucuronide form. However, the clearances and distributions of BPS and BPS-G were lower than those of the corresponding unconjugated and glucuroconjugated forms of BPA.

Inefficient UGT-conjugation of adrenal 11β-hydroxyandrostenedione metabolites highlights C11-oxy C19 steroids as the predominant androgens in prostate cancer

Although the adrenal C19 steroids, androstenedione and testosterone, contribute to prostate cancer (PCa) progression the full complement of adrenal androgens, including the C11-oxy C19 steroids, 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) and their androgenic metabolites, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) have, to date, not been considered. This study investigated the contribution of 11OHA4 and 11OHT to the pool of active androgens in the prostate. Steroid profiles were determined in LNCaP, C4-2B and VCaP cell models, in PCa tissue, and in plasma focussing on the inactivation, reactivation and glucuronidation of 11OHA4, 11OHT and their downstream products using ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). The C11-oxy C19 steroids were the predominant steroids with the production of 11KT and 11KDHT in prostate cell models identifying 11β-hydroxysteroid dehydrogenase type 2 activity. Active:inactive steroid ratios indicated efficient inactivation of dihydrotestosterone (DHT) and 11KDHT by 3α-hydroxysteroid dehydrogenases, while the reactivation of DHT by retinol-like dehydrogenases was greater than the reactivation of 11KDHT. In PCa tissue, inactive C11-oxy C19 steroids ranged from 27 to 30 ng/g, whereas inactive C19 steroids were below 1 ng/g. Steroid glucuronidation was impeded: in VCaP cells, the C11-oxy C19 steroids were unconjugated and the C19 steroids fully conjugated; in C4-2B cells, all steroids were unconjugated, except for DHT of which 50% was conjugated; in LNCaP cells only androsterone, 11KT and 11β-hydroxyandrosterone were unconjugated. In PCa patients’ plasma 11KDHT was present only in the unconjugated form, with 11KT also predominantly unconjugated (90–95%). Even though plasma and tissue sample numbers were limited, this study serves to demonstrate the abundance of C11-oxy C19 steroids, with notable differences in their metabolism, dictated by steroidogenic enzymes and hampered conjugation, affecting active androgen levels. Larger cohorts are required to analyse profiles in modulated metabolic pathways, in order to shed light on treatment outcomes. The C11-oxy C19 steroids are involved in PCa, with impeded glucuronidation in PCa ascribing a dominant role to these steroids in disease progression.

Methods for efficient analysis of tocopherols, tocotrienols and their metabolites in animal samples with HPLC-EC

Tocopherols and tocotrienols, collectively known as vitamin E, have received a great deal of attention because of their interesting biological activities. In the present study, we reexamined and improved previous methods of sample preparation and the conditions of high-performance liquid chromatography for more accurate quantification of tocopherols, tocotrienols and their major chain-degradation metabolites. For the analysis of serum tocopherols/tocotrienols, we reconfirmed our method of mixing serum with ethanol followed by hexane extraction. For the analysis of tissue samples, we improved our methods by extracting tocopherols/tocotrienols directly from tissue homogenate with hexane. For the analysis of total amounts (conjugated and unconjugated forms) of side-chain degradation metabolites, the samples need to be deconjugated by incubating with β-glucuronidase and sulfatase; serum samples can be directly used for the incubation, whereas for tissue homogenates a pre-deproteination step is needed. The present methods are sensitive, convenient and are suitable for the determination of different forms of vitamin E and their metabolites in animal and human studies. Results from the analysis of serum, liver, kidney, lung and urine samples from mice that had been treated with mixtures of tocotrienols and tocopherols are presented as examples.

Transhepatic bile acid kinetics in pigs and humans

Bile acids (BAs) play a key role in lipid uptake and metabolic signalling in different organs including gut, liver, muscle and brown adipose tissue. Portal and peripheral plasma BA concentrations increase after a meal. However, the exact kinetics of postprandial BA metabolism have never been described in great detail. We used a conscious porcine model to investigate postprandial plasma concentrations and transorgan fluxes of BAs, glucose and insulin using the para-aminohippuric acid dilution method. Eleven pigs with intravascular catheters received a standard mixed-meal while blood was sampled from different veins such as the portal vein, abdominal aorta and hepatic vein. To translate the data to humans, fasted venous and portal blood was sampled from non-diabetic obese patients during gastric by-pass surgery. The majority of the plasma bile acid pool and postprandial response consisted of glycine-conjugated forms of primary bile acids. Conjugated bile acids were more efficiently cleared by the liver than unconjugated forms. The timing and size of the postprandial response showed large interindividual variability for bile acids compared to glucose and insulin. The liver selectively extracts most BAs and BAs with highest affinity for the most important metabolic BA receptor, TGR5, are typically low in both porcine and human peripheral circulation. Our findings raise questions about the magnitude of a peripheral TGR5 signal and its ultimate clinical application.

Exposure to di-2-ethylhexyl terephthalate in a convenience sample of U.S. adults from 2000 to 2016

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of di-2-ethylhexyl phthalate (DEHP), is a plasticizer used in a variety of commercial applications, but data on Americans' exposure to DEHTP do not exist. We investigated the exposure to DEHTP in a convenience group of U.S. adults by analyzing urine collected anonymously in 2000 (N = 44), 2009 (N = 61), 2011 (N = 81), 2013 (N = 92), and 2016 (N = 149) for two major DEHTP oxidative metabolites: mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) and mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP). For comparison, we also quantified the analogous DEHP metabolites mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP) and mono-2-ethyl-5-carboxypentyl phthalate (MECPP). We detected MECPTP, MEHHP, and MECPP in all samples collected in 2016 with geometric means of 13.1, 4.1, and 6.7ng/mL, respectively; we detected MEHHTP in 91% of the samples (geometric mean = 3.1ng/mL). Concentrations of MECPTP correlated well with those of MEHHTP (R 2 = 0.8, p < 0.001), but did not significantly correlate with those of MEHHP (p > 0.05) suggesting different sources of exposure to DEHP and DEHTP. We also evaluated the fraction of the metabolites eliminated in their free (i.e., unconjugated) form. The median percent of unconjugated species was lower for the DEHP metabolites (MECPP [45.5%], MEHHP [1.9%]) compared to the DEHTP metabolites (MECPTP [98.8%], MEHHTP [21.2%]). Contrary to the downward trend from 2000 to 2016 in urinary concentrations of MEHHP and MECPP, we observed an upward trend for MEHHTP and MECPTP. These preliminary data suggest that exposure to DEHTP may be on the rise. Nevertheless, general population exposure data using MEHHTP and MECPTP as exposure biomarkers would increase our understanding of exposure to DEHTP, one of the known DEHP alternatives.

Reduced responses to glutamate receptor agonists follow loss of astrocytes and astroglial glutamate markers in the nucleus tractus solitarii.

Saporin (SAP) or SAP conjugates injected into the nucleus tractus solitarii (NTS) of rats kill astrocytes. When injected in its unconjugated form, SAP produces no demonstrable loss of or damage to local neurons. However bilateral injections of SAP significantly attenuate responses to activation of baroreceptor reflexes that are mediated by transmission of signals through glutamate receptors in the NTS. We tested the hypothesis that SAP would reduce cardiovascular responses to activation of NTS glutamate receptors despite its recognized ability to spare local neurons while killing local astrocytes. In animals treated with SAP and SAP conjugates or, as a control, with the toxin 6‐hydroxydopamine (6‐OHDA), we sought to determine if dose‐related changes of arterial pressure (AP) or heart rate (HR) in response to injection into NTS of N‐methyl‐d‐aspartate (NMDA) or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) were attenuated. Also we quantified changes in immunoreactivity (IR) for EAAT2, EAAC1, and VGluT2 in NTS after SAP and SAP conjugates. Our earlier studies showed that IR for NMDA and AMPA receptors was not changed after injection of SAP. We found that EAAT2 and EAAC1, both found in astrocytes, were reduced by SAP or its conjugates but not by 6‐OHDA. In contrast, VGluT2‐IR was increased by SAP or conjugates but not by 6‐OHDA. AP and HR responses to NMDA and AMPA were attenuated after SAP and SAP conjugate injection but not after 6‐OHDA. Results of this study are consistent with others that have shown interactions between astroglia and neurons in synaptic transmission mediated by glutamate receptor activation in the NTS.

3,4-Seco-12α-hydroxy-5β-cholan-3,4,24-trioic Acid, a Novel Secondary Bile Acid: Isolation from the Bile of the Common Ringtail Possum (Pseudocheirus peregrinus) and Chemical Synthesis

The major bile acids present in gallbladder bile of the common ringtail possum (Pseudocheirus peregrinus), an Australian marsupial, were isolated by preparative HPLC and identified by NMR and by comparison with synthetic standards. The major compound present (52%) was 3α,12α-dihydroxy-7-oxo-5β-cholan-24-oic acid (7-oxodeoxycholic acid), about three fourths conjugated with taurine. Also present was 3α,7β,12α-trihydroxy-5β-cholan- 24-oic acid (20%; ursocholic acid) largely in unconjugated form. In addition, 3,4-seco-12α-hydroxy-5β-cholan-3,4,24- trioic acid was present in unconjugated form and constituted 8% of biliary bile acids. Proof of the structure of this novel 3,4-seco acid was obtained by its chemical synthesis from deoxycholic acid via an intermediary 3β,4β-dihydroxy derivative that was then oxidatively cleaved with sodium periodate. As all primary bile acids have a hydroxyl or oxo substituent at C-7, the absence of such in the seco-bile acid suggests that it is a secondary bile acid, synthesized by bacterial enzymes present in the intestine.

UHPLC-MS/MS bioanalysis of urinary DHEA, cortisone and their hydroxylated metabolites as potential biomarkers for CYP3A-mediated drug-drug interactions.

A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7β-hydroxy-DHEA, cortisone and 6β-hydroxycortisone as potential biomarkers to predict CYP3A activity. A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7β-hydroxy-DHEA. This assay was successfully applied to a pilot clinical study. It is also suitable for future drug-drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

Role of steroid sulfatase in steroid homeostasis and characterization of the sulfated steroid pathway: Evidence from steroid sulfatase deficiency

The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol.

Disposition and Pharmacokinetics of a GalNAc3-Conjugated Antisense Oligonucleotide Targeting Human Lipoprotein (a) in Monkeys.

Triantennary N-acetyl galactosamine (GalNAc3)-conjugated antisense oligonucleotides (ASOs) have greatly improved potency due to receptor-mediated uptake into hepatocyte. The disposition and pharmacokinetics of ISIS 681257, a GalNAc3-conjugated ASO, were studied in monkeys. Following subcutaneous (SC) injection, ISIS 681257 was rapidly absorbed into the systemic circulation, with peak plasma levels observed within hours after dosing. After reaching Cmax, plasma concentrations rapidly declined in a multiexponential manner and were characterized by a dominant initial rapid distribution phase in which drug transferred to tissues from circulation, followed by a much slower terminal elimination phase (half-life of 4 weeks). Intact ISIS 681257 is the major full-length oligonucleotide species in plasma (≥70%). In tissues, the conjugated-GalNAc sugar moiety was rapidly metabolized, leaving the fully unconjugated form as the only full-length oligonucleotide detected at 48 h after dosing. Unconjugated ISIS 681257 cleared slowly from tissues with a half-life of 4 weeks. ISIS 681257 was highly bound to plasma proteins (>97% bound), which limited its urinary excretion. Disposition of ISIS 681257 in plasma and liver appeared nonlinear over the 1-40 mg/kg dose range studied. The plasma and liver tissue concentration data were well described by a population based mixed-effects modeling approach with Michaelis-Menten uptake from plasma to liver. Safety data from the study and the good exposure, as well as the extended half-life of the unconjugated ASO in the liver, support further development and less frequent dosing in Phase I clinical study.

Postprandial Plasma Concentrations of Individual Bile Acids and FGF-19 in Patients With Type 2 Diabetes.

Bile acids regulate lipid and carbohydrate metabolism by interaction with membrane or intracellular proteins including the nuclear farnesoid X receptor (FXR). Postprandial activation of ileal FXR leads to secretion of fibroblast growth factor 19 (FGF-19), a gut hormone that may be implicated in postprandial glucose metabolism. To describe postprandial plasma concentrations of 12 individual bile acids and FGF-19 in patients with type 2 diabetes (T2D) and healthy controls. Descriptive study, performed at the Center for Diabetes Research, Gentofte Hospital, Hellerup, Denmark. Fifteen patients with T2D and 15 healthy matched controls with normal glucose tolerance. A 75-g oral glucose tolerance test and three isocaloric and isovolemic liquid meals with low, medium, and high fat content, respectively. Bile acid and FGF-19 concentrations. Postprandial total bile acid concentrations increased with increasing meal fat content (P < .05), peaked after 1-2 hours, and were higher in T2D patients vs controls (oral glucose tolerance test, low and medium fat meals, P < .05; high fat meal, P = .30). Differences reflected mainly unconjugated and glycine-conjugated forms of deoxycholic acid (DCA) and to a lesser extent cholic acid (CA) and ursodeoxycholic acid (UDCA), whereas chenodeoxycholic acid (CDCA) concentrations were comparable in the two groups. FGF-19 concentrations tended to be lower in T2D patients vs controls, but differences were not statistically significant due to considerable variation. Postprandial plasma patterns of bile acids with FXR agonistic properties (CDCA, DCA, and CA) and FXR antagonistic properties (UDCA) in T2D patients support the notion of a "T2D-bile acid-FGF-19" phenotype with possible pathophysiological implications.

SFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo.

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Analysis of Cannabinoids and Their Metabolites in Human Urine.

Biologically monitoring marijuana exposure from active and passive use requires both a wide linear range and sensitive detection. We have developed and validated a multifunctional method using ultrahigh performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) for analysis of urinary Δ9-tetrahydrocannabinol (THC), cannabidiol and cannabinol, and two major metabolites of THC, 11-nor-9-carboxy-THC and 11-hydroxy-THC, in active users and particularly in people exposed to secondhand marijuana smoke (SHMS). The method used positive electrospray ionization (ESI) mode to reach the sensitivity needed to detect trace SHMS exposure with limits of detection (LOD) ranging from 0.002 to 0.008 nanograms per milliliter (ng/mL) and 0.005 to 0.017 ng/mL for "free" (unconjugated forms) and "total" (unconjugated plus conjugated forms) measurements, respectively. These LODs were approximately 10-100 times more sensitive than those reported in the literature. To reduce or avoid time-consuming repetitive sample preparation and analysis, the method simultaneously monitored multiple reaction monitoring transitions in negative ESI mode to quantify high analyte levels typically found in the urine of active marijuana users (linear dynamic range of 12.5-800 ng/mL). The validation results indicated this method was accurate (average inter/intra-day bias, <10%), precise (inter/intra-day imprecision, <10%), and fast (6 min run time). In addition, sample preparation throughput was greatly improved using an automation liquid-handling system, meeting the needs for potential large-scale population studies.

A Synthetic Disaccharide Analogue from Neisseria meningitidis A Capsular Polysaccharide Stimulates Immune Cell Responses and Induces Immunoglobulin G (IgG) Production in Mice When Protein-Conjugated

Some new phosphonoester-linked oligomers, stabilized analogues of the corresponding phosphate-bridged oligomers of Neisseria meningitidis A (MenA) capsular polysaccharide (CPS), were conjugated to human serum albumin (HSA), as a protein carrier model, and studied for immunological activities. We determined (i) in vitro, their biocompatibility (CAM test) and activity in inducing both T cell proliferation (CFSE method) and IL-2 release (ELISA), and (ii) in vivo, their ability to stimulate specific IgG antibody production (ELISA). All HSA-conjugated compounds induce T cell proliferation (40% of proliferation at 10(2) μM), whereas only the phosphonodisaccharide was effective (28% of proliferation at 10(2) μM) among the unconjugated forms. IL-2 release confirmed these results. In addition, the HSA-conjugated showed in vivo the capacity of eliciting the production of specific IgG antibodies. In conclusion, we obtained novel biocompatible, water-stable, and immunoactive MenA CPS analogues. A short disaccharide fragment showed the unusual behavior of triggering T cell proliferation in vitro.

Abstract P6-02-03: SUMO Inhibitors affect tumorigenesis of novel breast cancer xenograft model

Abstract A novel basal breast cancer cell line IOWA-1T was derived from chemotherapy resistant locally advanced breast cancer tumor. The cells rapidly form large, skin-eroding xenografts in nude mice. The SUMO inhibitor anacardic acid (AA) effectively cleared CD44+/hi/CD24-/low cancer stem cell (CSC) population in IOWA-1T and BT-20 basal cancer cell lines and delayed tumor outgrowth of basal cancer xenografts. The effect of SUMO inhibitors to clear the CSC population was dependent upon the SUMO unconjugated form of TFAP2A (Bogachek MV et al, Cancer Cell, 2014). Herein we show that tumors that eventually form from IOWA-1T xenografts in mice treated with AA are not capable of developing secondary xenografts, confirming eradication of the CSC population by SUMO inhibitors. As further mechanistic evidence for the SUMO pathway, transient knockdown of UBC9 and PIAS1 SUMOylation enzymes repressed CD44 expression and increased tumor free and overall survival in mice inoculated with IOWA-1T xenografts. Furthermore, CD44 downregulation was demonstrated in IOWA-1T cells after treatment in vitro with UBC9 inhibitor PYR-41 and PIAS1 inhibitor NSC-207895. Overall survival of mice with IOWA-1T xenografts was increased to 43±0.5 and 39±2 days with PYR-41 and NSC-207895 i.p. injections, respectively, compared to a vehicle treated control group 33±1 days (p&amp;lt;0.05). By contrast, doxorubicin treatment was not able to extend survival of mice with IOWA-1T xenografts. These findings establish the class of SUMO inhibitors as potential therapeutic drugs that eliminate the breast CSC population and may be effective in basal breast cancer cases resistant to conventional chemotherapy. Citation Format: Maria V Bogachek, Jung M Park, James P De Andrade, Mikhail V Kulak, Jeffrey R White, Tong Wu, Philip M Spanheimer, George W Woodfield, Thomas B Bair, Alicia K Olivier, Ronald J Weigel. SUMO Inhibitors affect tumorigenesis of novel breast cancer xenograft model [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-03.

PEGylation of the peptide Bac7(1–35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity

The proline-rich antibacterial peptide Bac7(1–35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1–35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1–35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials.

Sensing Properties of Long Period Fiber Grating Coated by Silver Film

In long period fiber gratings (LPFGs), the fiber modes guided by the core layer can excite surface plasmon resonance (SPR) in the metallic thin-film coated on the cylindrical surface of fibers. In this letter, the LPFG-based fiber optic SPR sensor is analyzed numerically by unconjugated form of coupled mode method. According to the mode analysis, the EH modes can couple with surface plasmon mode and are suitable for refractive index sensing application. In wavelength interrogation, the transmission dip caused by SPR is sensitive to the environmental refractive index. The sensitivity of the sensor achieves 1660 nm/RIU in the aqueous solution.

Improved profiling of estrogen metabolites by orbitrap LC/MS

Estrogen metabolites are important biomarkers to evaluate cancer risks and metabolic diseases. Due to their low physiological levels, a sensitive and accurate method is required, especially for the quantitation of unconjugated forms of endogenous steroids and their metabolites in humans. Here, we evaluated various derivatives of estrogens for improved analysis by orbitrap LC/MS in human serum samples. A new chemical derivatization reagent was applied modifying phenolic steroids to form 1-methylimidazole-2-sulfonyl adducts. The method significantly improves the sensitivity 2–100 fold by full scan MS and targeted selected ion monitoring MS over other derivatization methods including, dansyl, picolinoyl, and pyridine-3-sulfonyl products.