An eDNA assay for Irish Petromyzon marinus and Salmo trutta and field validation in running water.

This pilot study presents an environmental DNA (eDNA) assay for sea lamprey Petromyzon marinus and brown trout Salmo trutta, two species of economic and conservation importance in the Republic of Ireland. The results demonstrate the effectiveness of eDNA for assessing presence of low-abundance taxa (here, P. marinus) for environmental managers, and they highlight the potential for assessing relative abundance of rare or invasive freshwater species.

Deciphering the tumour immune microenvironment cell by cell.

Immune checkpoint inhibitors (ICIs) have rejuvenated therapeutic approaches in oncology. Although responses tend to be durable, response rates vary in many cancer types. Thus, the identification and validation of predictive biomarkers is a key clinical priority, the answer to which is likely to lie in the tumour microenvironment (TME). A wealth of data demonstrates the huge impact of the TME on ICI response and resistance. However, these data also reveal the complexity of the TME composition including the spatiotemporal interactions between different cell types and their dynamic changes in response to ICIs. Here, we briefly review some of the modalities that sculpt the TME, in particular the metabolic milieu, hypoxia and the role of cancer-associated fibroblasts. We then discuss recent approaches to dissect the TME with a focus on single-cell RNA sequencing, spatial transcriptomics and spatial proteomics. We also discuss some of the clinically relevant findings these multi-modal analyses have yielded.

Assisted reproductive technology treatment outcomes.

Information on the outcomes of ART treatments in Ireland is not readily available to Irish practitioners. The data for hospital affiliated clinics has been made available for many years and is included in the hospital reports. We present a 10-year analysis of the Irish ART results voluntarily reported by six out of seven IVF clinics. The data was collected from published ESHRE reports and from results (2007-8) not yet published. Data collected included: number of clinics and ART cycles, female age, clinical and multiple pregnancy rates and treatment complications. The clinical pregnancy rate per embryo transfer was 31.7% for IVF and 29.8% for ICSI. The proportion of singleton, twin and triplet deliveries for IVF and ICSI combined was 75%, 23.35% and 1.64%. The rate of ovarian hyperstimulation was 0.8%. ART practice in Ireland is safe, effective and responsible. Financial and societal savings could result from the introduction of state funded IVF with compulsory eSET where recommended.

Cytochrome P450 2B6 516G-->T is associated with plasma concentrations of nevirapine at both 200 mg twice daily and 400 mg once daily in an ethnically diverse population.

OBJECTIVES: The aim of the study was to characterize the impact of the cytochrome P450 2B6 (CYP2B6), CYP3A4, CYP3A5 and ATP-binding cassette sub-family B member 1 (ABCB1) polymorphisms on nevirapine plasma concentrations. METHODS: A total of 104 patients (82% male; 26% non-Caucasian) were genotyped for eight single nucleotide polymorphisms at four loci (CYP2B6, CYP3A4, CYP3A5 and MDR1). Nevirapine plasma concentrations were determined using high-performance liquid chromatography. RESULTS: Non-Caucasian ethnicity [5609 ng/mL (n=27) for non-Caucasians vs. 3771 ng/mL (n=77) for Caucasians; P<0.0001] and CYP2B6 516G-->T [GG, 3574 ng/mL (n=50); GT, 4634 ng/mL (n=50); TT, 8170 ng/mL (n=4); P(analysis of variance (anova))=0.001] were significantly associated with a higher nevirapine trough concentration (C(trough)). The latter association was maintained with both 200 mg twice daily (bid) and 400 mg once daily (qd) dosing [GG, 3527 ng/mL (n=30); GT, 4525 ng/mL (n=32); TT, 7020 ng/mL (n=2); P(anova)=0.05 and GG, 3645 ng/mL (n=20); GT, 4861 ng/mL (n=17); TT, 9508 ng/mL (n=2); P(anova)=0.01, respectively]. In a multivariable analysis, CYP2B6 516G-->T and non-Caucasian ethnicity remained significant predictors of nevirapine C(trough) but CYP2B6 516G-->T homozygosity had the greatest effect (108% higher, 46% higher). No associations were found between nevirapine C(trough) and the remaining polymorphisms. CONCLUSION: In this population, both non-Caucasian ethnicity and carriage of the variant allele of CYP2B6 516G-->T were significant predictors of nevirapine C(trough). The association between CYP2B6 516G-->T and higher plasma nevirapine exposure was maintained at both bid and qd dosing.

Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond.

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

Widespread use of herbal medicines by people living with human immunodeficiency virus and contamination of herbal medicines with antiretrovirals in Nigeria.

Herbal medication use amongst people living with human immunodeficiency virus (PLWH) is widespread and understudied. This study aimed to evaluate the prevalence of herbal medicine use amongst PLWH and possible contamination with antiretrovirals (ARVs). Countrywide collection of herbal samples sold by street vendors in Nigeria for the following indications: human immunodeficiency virus (HIV), acquired immune deficiency syndrome, fever and general weakness. Samples were screened using a validated liquid chromatography-mass spectrometry/mass spectrometry method for the presence of the following ARVs: efavirenz, nevirapine, lopinavir, darunavir, ritonavir, atazanavir, emtricitabine, tenofovir and lamivudine. A survey was conducted among 742 PLWH attending four HIV clinics in Nigeria. Data were collected using a structured questionnaire and analysed using IBM SPSS statistics version 22.0 (IBM Corp., 2013, Armond, NY). Of the 138 herbal medicines sampled, three (2%) contained detectable levels of tenofovir, emtricitabine and/or lamivudine. Additionally, of the 742 PLWH surveyed, 310 (41.8%) reported herbal medicine use. Among the users, 191 (61.6%) started taking herbals after commencing HIV therapy while herbal medicine use preceded ARVs treatment in 119 (38.4%) PLWH. We found herbal use to be widespread among PLWH in Nigeria, with increasing use after commencing ARV. Three herbal preparations were also found to contain detectable levels of ARVs. This is a concern and should be studied widely across the region and countries where herbal medicine use is prevalent and poorly regulated.

Molecular genetics of acute promyelocytic leukemia.

The remarkable success of retinoic acid in the treatment of acute promyelocytic leukemias and the subsequent discovery that mutant forms of a retinoid acid receptor (RARalpha) are invariably associated with this disease has generated considerable interest among both clinicians and basic scientists. Studies both in cell culture and in transgenic animals suggest that mutant RARs interfere with normal retinoid-mediated transactivation and granulocytic differentiation. More recently, a pivotal link between transcriptional silencing, the oncogenic functions of RAR mutants, and hormonal responses in APL patients has been established. These studies have greatly advanced our understanding of the molecular changes involved in leukemogenesis, have helped to reveal new aspects of cellular differentiation, and might lead to improved treatment strategies for human leukemias.

The frequency and impact of restless legs syndrome in primary care.

This study examined the prevalence and impact of moderate to severe RLS (MS-RLS) in primary care patients in Ireland. Patients completed a screening questionnaire and those with symptoms suggestive of MS-RLS underwent a diagnostic interview. Patients diagnosed with MS-RLS completed quality of life and sleep assessment questionnaires, and their medical records were examined. Of 2628 patients screened for RLS, 74 (2.8%, 95% confidence interval 2.2%-3.5%) were ultimately diagnosed at interview as having MS-RLS. These patients reported significant impact on sleep and quality of life; 24 (32.4%) had consulted a health care professional about their RLS symptoms but only 4 (16.7%) were diagnosed with RLS. Ten (13.5%) MS-RLS patients were taking inappropriate medicines to try to relieve their symptoms. Clinically significant RLS is common in Irish general practice and has a significant effect on sleep and quality of life. Nevertheless, the condition often goes undiagnosed.

Cervical screening during pregnancy.

UNLABELLED: A retrospective study done to assess the efficiency of cervical screening in the form of smear testing in pregnant population. We selected one hundred women who had undergone cervical screening during booking visit in one particular unit. Results of tests along with datas regarding age, parity and previous smear history was compiled. Results were compared with Western Health Board figures. 58% smears were normal, 6 % abnormal and 36% unsatisfactory. Although incidence unsatisfactory smears were very high, but incidence of abnormal cytology matched with standard. CONCLUSION: Pregnant women are no more likely than the general population to have a frankly abnormal smear. Despite its limitations we recommend antenatal screening does have a role in the absence of National Cervical Screening Programme.

Human placental chorionic renin: production, purification and characterization.

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.

Prescribing medication in long-term dialysis units.

Little information is available regarding the current patterns of medication use in long-term dialysis centers. Therefore, we surveyed the medication records of 1,023 patients undergoing long-term dialysis therapy in 27 dialysis centers. The mean number of medications prescribed per patient was 7.7 +/- 0.54, increasing patient age, increasing duration of dialysis, in-center dialysis, and the presence of underlying diabetic and hypertensive nephropathy were associated with increased frequency of medication use. The use of multiple pharmacologic agents was associated with a high frequency of drug duplication (12%), potential dosage error (9%), potential significant drug interaction (15%), and use of contraindicated drugs (2.5%). A lack of individualization of the use of several pharmacologic agents was apparent. An extreme degree of center variability in drug use was also apparent. Periodic review of medication use should be undertaken in the long-term dialysis setting.

Pharmacokinetic and Pharmacodynamic Comparison of Once-Daily Efavirenz (400 mg vs. 600 mg) in Treatment-Naïve HIV-Infected Patients: Results of the ENCORE1 Study.

Daily efavirenz 400 mg (EFV400) was virologically noninferior to 600 mg (EFV600) at 48 weeks in treatment-naïve patients. We evaluated EFV400 and EFV600 pharmacokinetics (NONMEM v. 7.2), assessing patient demographics and genetic polymorphisms (CYP2B6, CYP2A6, CYP3A4, NR1I3) as covariates and explored relationships with efficacy (plasma HIV-RNA (pVL) <200 copies/mL) and safety outcomes at 48 weeks in 606 randomized ENCORE1 patients (female = 32%, African = 37%, Asian = 33%; EFV400 = 311, EFV600 = 295). CYP2B6 516G>T/983T>C/CYP2A6*9B/*17 and weight were associated with efavirenz CL/F. Exposure was significantly lower for EFV400 (geometric mean ratio, GMR; 90% confidence interval, CI: 0.73 (0.68-0.78)) but 97% (EFV400) and 98% (EFV600) of evaluable pVL was <200 copies/mL at 48 weeks (P = 0.802). Four of 20 patients with mid-dose concentrations <1.0 mg/L had pVL ≥200 copies/mL (EFV400 = 1; EFV600 = 3). Efavirenz exposure was similar between those with and without efavirenz-related side effects (GMR; 90% CI: 0.95 (0.88-1.02)). HIV suppression was comparable between doses despite significantly lower EFV400 exposure. Comprehensive evaluation of efavirenz pharmacokinetics/pharmacodynamics revealed important limitations in the accepted threshold concentration.

Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

Models of a heroin epidemic.

Heroin addiction may be considered an epidemic disease that is communicated by people who are addicted to the drug. It has been suggested that the most recent epidemic in the United States had its peak incidence in 1969. The age of heroin addicts entering treatment has increased systematically from 1973 to 1976 at a rate of less than one year of age per calendar year. This pattern is consistent with a moderate decline in a national heroin epidemic or a geographical migration of the epidemic from more to less populated areas. There are also seasonal trends in the age of admission to treatment.

Prevention in mental health: a controlled study.

The authors conducted a controlled study in which families who had experienced the sudden death of a family member were given crisis intervention services and compared at follow-up with two untreated control groups. Results did not support the hypothesis that such services decrease the risk of psychiatric illness, disturbed family functioning, or increased social cost to the families. The authors suggest that environmental and social systems factors and individual variables are powerful predictors of outcome in bereavement.

Renin inhibitors. Dipeptide analogues of angiotensinogen utilizing a dihydroxyethylene transition-state mimic at the scissile bond to impart greater inhibitory potency.

The synthesis of diol-containing renin inhibitors has revealed that a simple vicinal diol functionality corresponding to the scissile Leu-Val bond in human angiotensinogen is capable of imparting inhibitory activity at a comparable or higher level than either the corresponding aldehyde or hydroxymethyl functionality (compare inhibitors 2a-c or 3a-c). This finding has led to the further optimization of a series of small transition-state analogue inhibitors by the inclusion of a second hydroxyl group in the Leu-Val surrogate to give compounds that inhibited human renin in the 200-700-pM range (e.g. 43, 45, 63, 66). The magnitude of effect of the second hydroxyl group on potency is not only dictated by the absolute stereochemistry of the diol but also by the side chain of the P1 residue. Molecular modeling of the diol-containing inhibitors suggests that one of the hydroxyl groups hydrogen bonds to Asp 32 and Asp 215, while the second hydrogen bonds to Asp 215. These diol inhibitors are extremely selective for human renin over the related enzymes cathepsin D, pepsin, and gastricsin. At high concentrations, compounds containing a leucine or phenylalanine rather than a histidine at the P2 position gave only minor amounts of inhibition of the other enzymes. Inhibitor 43 suppressed plasma renin activity completely and lowered mean blood pressure in monkeys after both intravenous and intraduodenal administration, but the blood pressure drop lasted less than 1 h. Monitoring the blood levels of 43 by enzyme inhibition assay after intraduodenal administration to monkeys or oral administration to rats revealed low absorption and rapid clearance. While intratracheal administration to dogs gave approximately 50% bioavailability, rapid clearance was still a problem. After examination of inhibitor 45 in a sensitive primate model in which monkeys were rendered both hypertensive and hyperreninemic, the effects on lowering systolic but not diastolic pressure were apparent even after 22 h postdosing. Details on the synthesis, in vitro structure-activity relationships, molecular modeling, in vivo activity, and metabolism of these inhibitors are described.

Renin inhibitors based on dipeptide analogues. Incorporation of the hydroxyethylene isostere at the P2/P3 sites.

The synthesis of a series of renin inhibitors in which the P2 and P3 amino acids are replaced with the hydroxyethylene dipeptide isostere is reported. In vitro evaluation of the inhibitors has revealed that this isostere is an acceptable amide-bond replacement in which activity is maintained and stability is enhanced. Structure-activity relationships of this series resemble but do not parallel those of the corresponding dipeptide-containing inhibitors.

Studies directed toward the design of orally active renin inhibitors. 1. Some factors influencing the absorption of small peptides.

A systematic evaluation of structure-absorption relationships using a high throughput intraduodenal rat screening model has led to the delineation of a set of structural parameters that appear to govern bioavailability in a series of peptide-based renin inhibitors. Optimum structures, exemplified by 25 and 41, incorporated a single, solubilizing substituent at the C- or N-terminus combined with a lipophilic P2-site residue. Both inhibitors gave unprecedented plasma drug levels upon intraduodenal administration to monkeys, and the calculated bioavailability for 41 (14 +/- 4%) is the highest reported for any peptidic renin inhibitor.

C-terminal modifications of nonpeptide renin inhibitors: improved oral bioavailability via modification of physicochemical properties.

We describe the development of a series of soluble, potent, and bioavailable nonpeptide renin inhibitors. These inhibitors derived from a series of novel nonpeptide renin inhibitors which were recently identified in our laboratories, by alteration of the nature of the C-terminus (P2') of the molecules. Introduction of basic substituents into modified hydroxyethylene dipeptide isosteres gave inhibitors with improved solubility as well as improved potency against human plasma renin. In addition, these modifications produced inhibitors which displayed markedly improved intraduodenal bioavailability in both the ferret and cynomolgus monkey. We also present data which demonstrate excellent efficacy in the monkey for A-74273 (65), with an intraduodenal bioavailability of 16 +/- 4% in the monkey, compared to 1.7 +/- 0.5% for the dipeptide renin inhibitor enalkiren (A-64662, 75). A-74273 is an example of a nonpeptide inhibitor which possesses a good balance of the desirable properties of potency, solubility, and lipophilicity and which is well absorbed into the intestine.