Determination of steroid estrogens in wastewater by high performance liquid chromatography-tandem mass spectrometry

This paper discusses the requirement for, and presents an analytical procedure for, the determination of four unconjugated steroid hormones and a conjugated steroid (estrone-3-sulfate) in wastewaters. The method quantifies the steroids by LC/MS/MS following solid phase extraction and a two stage clean-up procedure. Samples were extracted using C18 cartridges and eluates were then purified by gel permeation chromatography, followed by a further clean-up step on an aminopropyl cartridge. The limits of detection achieved were 0.2 ng l-1 for estriol, 17β-estradiol and 17α-ethinylestradiol, and 0.1 ng l-1 for estrone and the conjugate. The robustness of the method was demonstrated by achieving recoveries of >83% for all steroids in settled sewage and final effluent samples with relative standard deviations of 0.5 - 12%. The method was used to analyse a range of samples from a wastewater treatment works in south east England which demonstrated a >80% removal for estrone, estradiol and estriol with little impact on concentrations of ethinylestradiol or the conjugate

Inhibition of hepatitis C virus RNA replication by ISG15 does not require its conjugation to protein substrates by the HERC5 E3 ligase

Chronic infection of the liver by hepatitis C virus (HCV) induces a range of host factors including IFN-stimulated genes such as ISG15. ISG15 functions as an antiviral factor that limits virus replication. Previous studies have suggested that ISG15 could influence HCV replication in both a positive and a negative manner. In this report, we determined the effect of ISG15 on HCV RNA replication in two independent cell lines that support viral genome synthesis by inhibiting ISG15 expression through small interfering RNA, short-hairpin RNA and CRISPR/Cas9 gene knockout approaches. Our results demonstrated that ISG15 impairs HCV RNA replication in both the presence and absence of IFN stimulation, consistent with an antiviral role for ISG15 during HCV infection. ISG15 conjugation to protein substrates typically requires the E3 ligase, HERC5. Our results showed that the inhibitory effect of ISG15 on HCV RNA replication does not require its conjugation to substrates by HERC5

Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans

Successful Pregnancy Outcome In Maternal Crigler Najjar Syndrome Type II.

Estimated incidence of Crigler-Najjar syndrome(CNS) is 1 case per 1,000,000 births(1 million). The overall prevalence of CN syndrome is unknown, with only several hundred people reported to have this disease. It is interestingly very rare to encounter a pregnant adult women with congenital jaundice. Pregnancy in CN type II patients is a diagnostic and a therapeutic challenge because of the high risk of bilirubin encephalopathy with serious neurological damage as life-threatening complications for the fetus. To date 8 pregnancy outcome have been reported from 5 women and we report the6 woman with a successful 9 th pregnancy outcome. We have discussed detail history, presentation and management during pregnancy and care of the new born

First-trimester or second-trimester screening, or both, for Down's syndrome

BACKGROUND: It is uncertain how best to screen pregnant women for the presence of fetal Down's syndrome: to perform first-trimester screening, to perform second-trimester screening, or to use strategies incorporating measurements in both trimesters.METHODS: Women with singleton pregnancies underwent first-trimester combined screening (measurement of nuchal translucency, pregnancy-associated plasma protein A [PAPP-A], and the free beta subunit of human chorionic gonadotropin at 10 weeks 3 days through 13 weeks 6 days of gestation) and second-trimester quadruple screening (measurement of alpha-fetoprotein, total human chorionic gonadotropin, unconjugated estriol, and inhibin A at 15 through 18 weeks of gestation). We compared the results of stepwise sequential screening (risk results provided after each test), fully integrated screening (single risk result provided), and serum integrated screening (identical to fully integrated screening, but without nuchal translucency).RESULTS: First-trimester screening was performed in 38,167 patients; 117 had a fetus with Down's syndrome. At a 5 percent false positive rate, the rates of detection of Down's syndrome were as follows: with first-trimester combined screening, 87 percent, 85 percent, and 82 percent for measurements performed at 11, 12, and 13 weeks, respectively; with second-trimester quadruple screening, 81 percent; with stepwise sequential screening, 95 percent; with serum integrated screening, 88 percent; and with fully integrated screening with first-trimester measurements performed at 11 weeks, 96 percent. Paired comparisons found significant differences between the tests, except for the comparison between serum integrated screening and combined screening.CONCLUSIONS: First-trimester combined screening at 11 weeks of gestation is better than second-trimester quadruple screening but at 13 weeks has results similar to second-trimester quadruple screening. Both stepwise sequential screening and fully integrated screening have high rates of detection of Down's syndrome, with low false positive rates

Modifications at the C-Terminus To Improve Pyrrole−Imidazole Polyamide Activity in Cell Culture

Pyrrole−imidazole (Py-Im) hairpin polyamides are a class of small molecule DNA minor groove binding compounds that have been shown to modulate endogenous gene expression in cell culture. Gene regulation by polyamides requires efficient cellular uptake and nuclear localization properties for candidate compounds. To further optimize Py-Im polyamides for enhanced potency in cell culture, a focused library of polyamides possessing various modifications at the C-terminus was synthesized and tested. Comparison of polyamide biological activity in two cell lines revealed tolerance for structural modifications and agreement in activity trends between cell lines. The use of an oxime linkage between the polyamide and an aromatic functionality on the C-terminus resulted in a ~20-fold increase in the potency of polyamides targeted to the androgen response element (ARE) in LNCaP cells by measuring AR-activated PSA expression

Induction of fecal cholesterol excretion is not effective for the treatment of hyperbilirubinemia in Gunn rats

Background Unconjugated hyperbilirubinemia, a feature of neonatal jaundice or Crigler-Najjar syndrome, can lead to neurotoxicity and even death. We previously demonstrated that unconjugated bilirubin (UCB) can be eliminated via transintestinal excretion in Gunn rats, a model of unconjugated hyperbilirubinemia, and that this is stimulated by enhancing fecal fatty acid excretion. Since transintestinal excretion also occurs for cholesterol (TICE), we hypothesized that increasing fecal cholesterol excretion and/or TICE could also enhance fecal UCB disposal and subsequently lower plasma UCB concentrations. Methods To determine whether increasing fecal cholesterol excretion could ameliorate unconjugated hyperbilirubinemia, we treated hyperbilirubinemic Gunn rats with ezetimibe (EZE), an intestinal cholesterol absorption inhibitor, and/or a liver X receptor (LXR) and farnesoid X receptor (FXR) agonist (T0901317 (T09) and obeticholic acid (OCA), respectively), known to stimulate TICE. Results We found that EZE treatment alone or in combination with T09 or OCA increased fecal cholesterol disposal but did not lower plasma UCB levels. Conclusions These findings do not support a link between the regulation of transintestinal excretion of cholesterol and bilirubin. Furthermore, induction of fecal cholesterol excretion is not a potential therapy for unconjugated hyperbilirubinemia. Impact Increasing fecal cholesterol excretion is not effective to treat unconjugated hyperbilirubinemia. This is the first time a potential relation between transintestinal excretion of cholesterol and unconjugated bilirubin is investigated. Transintestinal excretion of cholesterol and unconjugated bilirubin do not seem to be quantitatively linked. Unlike intestinal fatty acids, cholesterol cannot "capture" unconjugated bilirubin to increase its excretion. These results add to our understanding of ways to improve and factors regulating unconjugated bilirubin disposal in hyperbilirubinemic conditions

Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

Background: The upper gastrointestinal tract plays a prominent role in human physiology as the primary site for enzymatic digestion and nutrient absorption, immune sampling, and drug uptake. Alterations to the small intestine microbiome have been implicated in various human diseases, such as non-alcoholic steatohepatitis and inflammatory bowel conditions. Yet, the physiological and functional roles of the small intestine microbiota in humans remain poorly characterized because of the complexities associated with its sampling. Rodent models are used extensively in microbiome research and enable the spatial, temporal, compositional, and functional interrogation of the gastrointestinal microbiota and its effects on the host physiology and disease phenotype. Classical, culture-based studies have documented that fecal microbial self-reinoculation (via coprophagy) affects the composition and abundance of microbes in the murine proximal gastrointestinal tract. This pervasive self-reinoculation behavior could be a particularly relevant study factor when investigating small intestine microbiota. Modern microbiome studies either do not take self-reinoculation into account, or assume that approaches such as single housing mice or housing on wire mesh floors eliminate it. These assumptions have not been rigorously tested with modern tools. Here, we used quantitative 16S rRNA gene amplicon sequencing, quantitative microbial functional gene content inference, and metabolomic analyses of bile acids to evaluate the effects of self-reinoculation on microbial loads, composition, and function in the murine upper gastrointestinal tract. Results: In coprophagic mice, continuous self-exposure to the fecal flora had substantial quantitative and qualitative effects on the upper gastrointestinal microbiome. These differences in microbial abundance and community composition were associated with an altered profile of the small intestine bile acid pool, and, importantly, could not be inferred from analyzing large intestine or stool samples. Overall, the patterns observed in the small intestine of non-coprophagic mice (reduced total microbial load, low abundance of anaerobic microbiota, and bile acids predominantly in the conjugated form) resemble those typically seen in the human small intestine. Conclusions: Future studies need to take self-reinoculation into account when using mouse models to evaluate gastrointestinal microbial colonization and function in relation to xenobiotic transformation and pharmacokinetics or in the context of physiological states and diseases linked to small intestine microbiome and to small intestine dysbiosis

Progeny, June 2013, Vol 29, no.1

This newsletter from The Department of Public Health about perinatal health care and statistics

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types