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Bacteriophages exert strong evolutionary pressure on their microbial hosts. In their lytic lifecycle, complete bacterial subpopulations are utilized as hosts for bacteriophage replication. However, during their lysogenic lifecycle, bacteriophages can integrate into the host chromosome and alter the host's genomic make-up, possibly resulting in evolutionary important adjustments. Not surprisingly, bacteria have evolved sophisticated immune systems to protect against phage infection. Streptococcus pyogenes isolates are frequently lysogenic and their prophages have been shown to be major contributors to the virulence of this pathogen. Most S. pyogenes phage research has focused on genomic prophages in relation to virulence, but little is known about the defensive arsenal of S. pyogenes against lytic phage infection. Here, we characterized Phage A1, an S. pyogenes bacteriophage, and investigated several mechanisms that S. pyogenes utilizes to protect itself against phage predation. We show that Phage A1 belongs to the Siphoviridae family and contains a circular double-stranded DNA genome that follows a modular organization described for other streptococcal phages. After infection, the Phage A1 genome can be detected in isolated S. pyogenes survivor strains, which enables the survival of the bacterial host and Phage A1 resistance. Furthermore, we demonstrate that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Lastly, we show that S. pyogenes produces membrane vesicles that bind to phages, thereby limiting the pool of phages available for infection. Altogether, this work provides novel insight into survival strategies employed by S. pyogenes to combat phage predation.

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

During drug discovery and development, the early identification of adverse effects is expected to reduce costly late-stage failures of candidate drugs. As risk/safety assessment takes place rather late during the development process and due to the limited ability of animal models to predict the human situation, modern unbiased high-dimensional biology readouts are sought, such as molecular signatures predictive for in vivo response using high-throughput cell-based assays. In this theoretical proof of concept, we provide findings of an in-depth exploration of a single chemical core structure. Via transcriptional profiling, we identified a subset of close analogues that commonly downregulate multiple tubulin genes across cellular contexts, suggesting possible spindle poison effects. Confirmation via a qualified toxicity assay (in vitro micronucleus test) and the identification of a characteristic aggregate-formation phenotype via exploratory high-content imaging validated the initial findings. SAR analysis triggered the synthesis of a new set of compounds and allowed us to extend the series showing the genotoxic effect. We demonstrate the potential to flag toxicity issues by utilizing data from exploratory experiments that are typically generated for target evaluation purposes during early drug discovery. We share our thoughts on how this approach may be incorporated into drug development strategies.

The structure-activity relationships for a series of heteroaryl urea inhibitors of fatty acid amide hydrolase (FAAH) are described. Members of this class of inhibitors have been shown to inactivate FAAH by covalent modification of an active site serine with subsequent release of an aromatic amine from the urea electrophile. Systematic Ames II testing guided the optimization of urea substituents by defining the structure-mutagenicity relationships for the released aromatic amine metabolites. Potent FAAH inhibitors were identified having heteroaryl amine leaving groups that were non-mutagenic in the Ames II assay., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Sixteen coded compounds were blind-tested at 4 laboratories using the recently described GADD45a-GFP genotoxicity assay. The compounds were chosen to include non-genotoxic compounds as well as weak and strong genotoxins. None of the compounds required metabolic activation in order to exhibit genotoxic effects. The participating laboratories included 2 global pharmaceutical companies, a global consumer goods company and the Gentronix laboratory in Manchester. Each compound was tested 4 times on different days following a protocol previously described. The tests were carried out after a 3-day training period from the parent lab (Manchester). Following the exclusion of data from tests with positive control failures and data series with 'spikes', 92% of assays gave the correct result: non-genotoxins giving negative results and genotoxins giving positive results. There were no randomly distributed problems suggesting that differences between the results from different sites reflected the use of different instruments, procedural differences and operator experience. In naïve operator laboratories the quality of data improved with operator practice. It was concluded that simple clarification of the protocol would provide the level of reliability required for widespread use of the assay in hazard assessment.

An international, multi-lab trial was conducted to evaluate a flow cytometry-based method for scoring micronuclei in mouse lymphoma L5178Y cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Mol. Mutagen. 47 (2006) 56-66]. A reference laboratory investigated the potential of six chemicals to induce micronuclei -- the genotoxicants mitomycin C (MMC), etoposide (ETOPO), and vinblastine (VB), and the non-genotoxicants sucrose (SUC), staurosporine (STS), and dexamethasone (DEX). The latter two non-genotoxicants were selected as extreme challenges to the assay because of their potent apoptogenic activity. Three collaborating laboratories were supplied with prototype In Vitro MicroFlow kits, and each was assigned one genotoxicant and one non-genotoxicant. Cells were treated continuously for 24h over a range of concentrations up to 5 mg/ml, or overtly cytotoxic concentrations. Micronuclei were scored via standard microscopy and flow cytometry. In addition to enumerating micronucleus frequencies, a cytotoxicity measurement that is simultaneously acquired with the flow cytometric micronucleus scoring procedure was evaluated (Flow-NBR). With this method, latex particles served as counting beads, and facilitated relative survival measurements that exclude the presence of dead/dying cells. For comparison purposes, additional cytotoxicity endpoints were measured, including several that are based on cell number, and others that reflect compromised membrane integrity, including dye permeability and/or phospholipid distribution. Key findings for this set of compounds include the following: (1) significant discrepancies in top concentration selection were found when cytotoxicity measurements were based on different methods, with the Flow-NBR approach tending to be the most sensitive, (2) both microscopy- and flow cytometry-based scoring methods detected concentration-dependent micronucleus formation for the three genotoxic agents studied, with good agreement between the reference laboratory and the collaborating laboratories, and (3) whereas flow cytometric analyses showed no significant increases for the non-genotoxicants when top concentration selection was based on Flow-NBR, significantly elevated micronucleus frequencies were observed for concentrations that were chosen based on less-sensitive cytotoxicity assays. Collectively, these results indicate that rapid assessment of genotoxicity can be accomplished with a relatively simple flow cytometric technique, and that the scoring system is transferable across laboratories. Furthermore, a concurrent assessment of cytotoxicity, Flow-NBR, may help reduce the occurrence of irrelevant positive results, as it may represent a more appropriate means for choosing top concentration levels. Finally, the data presented herein reinforce concerns about the manner in which cytotoxicity limits are described in guidance documents, since these recommendations tend to cite fixed cut-off values without reference to methodology.

Objective: To assess blood oxygen binding in calves with diarrhea., Animals: 22 dairy and 26 double-muscled calves with diarrhea, 31 healthy dairy calves and 37 healthy double-muscled calves., Procedure: Severity of disease, including the ability of affected calves to stand, was evaluated. Hydration and signs of depression were scored. Venous and arterial blood samples were collected, and 2,3-diphosphoglycerate, ATP, chloride, inorganic phosphate, lactate, pyruvate, total protein, albumin, and hemoglobin concentrations, and Hct, pH, Pco2, and PO2 were determined. Oxygen equilibrium curves (OEC) were constructed under standard conditions, and oxygen extraction ratios were calculated., Results: Recumbent calves of both breed-types were more dehydrated and had more severe signs of depression than ambulatory affected calves. In both breed-types, hemoglobin oxygen affinity was increased in calves with diarrhea, compared with healthy calves, as indicated by a decrease in standard partial oxygen pressure (P50). Diarrhea induced hypocapnia and hypothermia in the most severely affected calves, which counteracted the acidosis-induced right shift in arterial and venous OEC. Arterial and venous P50 were significantly less in double-muscled calves with diarrhea than healthy calves, whereas P50 for affected dairy calves were similar to those of healthy calves. Except in the most severely affected dairy calves, oxygen extraction ratio was significantly less in calves with diarrhea, compared with healthy calves., Conclusions and Clinical Relevance: Release of oxygen from blood may be impaired in calves with diarrhea, depending on the effect of the disease on certain blood biochemical variables.

The effect of ammonia on the cough response to citric acid and on substance P release from C-fibers involved in this reflex was assessed. For a period from one to four days, piglets were exposed, in an inhalation chamber, to ammonia at a concentration of 15 or 30 ppm. During exposure, cough induction tests were done every two days. Recovery of the cough reflex after ammonia exposure was also determined. In a separate group of piglets exposed for 2 days to 30 ppm ammonia, substance P content was determined in bronchial and tracheal lavage fluids and in the tracheal and bronchial mucosa. Ammonia (30 ppm) was found to inhibit coughing significantly (the cough frequency was reduced by 64%) after a two-day exposure. In animals exposed for 4 days to this ammonia concentration, the recovery ranged from 3 to 7 days (mean: 5 days). The same ammonia concentration also caused the substance P content to increase significantly in bronchoalveolar lavage fluid (to 432% of its initial value) and tracheal lavage fluid (to 149%) and to decrease significantly in the tracheal mucosa (-58%), however the content in bronchial mucosa was not significantly affected (-43%). Exposure to 15 ppm ammonia had no effect on the frequency of citric acid-induced coughing. In conclusion, ammonia inhibits citric acid-induced coughing in pigs at concentrations that can be detected in piggeries. This inhibitory effect may be related to substance-P depletion in C-fiber endings.

Objective: To assess in vivo blood oxygen binding in double-muscled calves and dairy calves with conventional muscle conformation., Animals: 58 dairy and 48 double-muscled calves., Procedure: Calves were classified as neonatal (24 hours old) or older calves (2 to 26 days old). Venous and arterial blood samples were collected, and hemoglobin concentration, pH, PCO2, and PO2 were determined. Blood oxygen equilibrium curves (OEC) under standard conditions were constructed, and the oxygen exchange fraction (OEF) and the amount of oxygen released at the tissue level by 100 ml of blood (OEF Vol%) were calculated., Results: In each breed, partial pressure of oxygen at 50% saturation of hemoglobin (P50) under standard conditions was significantly higher in older than in neonatal calves, indicating a right shift in OEC with age. Venous P50 was significantly lower in neonatal double-muscled calves than in neonatal dairy calves, but arterial and venous P50 were significantly higher in older double-muscled calves than in older dairy calves. In double-muscled, but not in dairy, calves, OEF was significantly higher in older than in neonatal calves. In neonatal calves, OEF Vol% was not significantly different between breeds, but OEF Vol% was significantly higher in older double-muscled calves than in older dairy calves., Conclusions and Clinical Relevance: The lower OEF in neonatal double-muscled calves, compared with dairy calves, could contribute to the higher sensitivity of double-muscled calves to hypoxia. Blood oxygen affinity decreased with age, but OEF and OEF Vol% were unchanged with age in dairy calves, whereas they increased with age in double-muscled calves.

Objective: To assess the effects of inhalation of feed flour dust and dustborne endotoxin on respiratory tracts of pigs., Animals: 29 healthy Belgian Landrace pigs., Procedure: Pigs housed in an environmental chamber were exposed for 6 days to feed flour dust (1 to 15 mg/m3) and dustborne endotoxins (50 to 2,500 ng/m3). Effects were evaluated by measuring albumin concentration, lactate dehydrogenase (LDH) activity, cell composition of nasal lavage (NL) and bronchoalveolar lavage (BAL) fluids and blood, and percentages of CD4+ and CD8+ T lymphocytes in blood and lavage fluids. Dustborne endotoxin was obtained by mixing endotoxins from Escherichia coli (serotype O127:B8) with feed flour before spraying the flour in the environmental chamber., Results: Exposure did not affect cell composition of NL fluid or blood. Total cell counts of BAL fluids were increased in all groups exposed to dust. Macrophage counts were increased in pigs exposed to inhalable dust concentrations as low as 4.4 mg/m3, and lymphocyte counts were increased in groups exposed to high dust concentrations. Percentages of CD4+ and CD8+ T lymphocytes in blood and lavage fluids were unchanged. In all dust-exposed groups, albumin content of BAL fluid was increased, whereas LDH activity was unaffected. Macrophage and lymphocyte infiltration and edema in the bronchi were identified by light microscopy. Effects attributable to E. coli endotoxin exposure were not identified., Conclusions: Inhalation of feed flour dust did not affect nasal mucosa but did induce bronchial airway inflammation. Dustborne endotoxins did not have effects attributable to endotoxin alone.

A new experimental setup was developed to expose pigs to dust and airborne endotoxins in an environmental chamber, at levels liable to be encountered in pig farm buildings. The following parameters were evaluated in a chamber containing two pigs of 10 kg body-weight: inhalable and respirable dust gravimetric concentrations were measured using area samplers and expressed as mg/m3. The respirable dust concentration was also measured using a "TM digital microP respirable dust-measuring instrument', which has been shown to give similar results to the gravimetric method. The endotoxin concentration was evaluated using the Limulus-assay and expressed as ng/m3 of air containing the inhalable or respirable dust or as ng/mg of inhalable and respirable dust. Feed flour dust was introduced into the chamber to obtain different concentrations of inhalable and respirable dust ranging from 3.62 to 76.66 mg/m3 and from 0.24 to 1.40 mg/m3, respectively. The endotoxin concentration was modulated by mixing the feed flour with Escherichia coli endotoxins before blowing it into the chamber. The endotoxin concentrations in the air containing inhalable or respirable dust ranged from 28.9 to 270.0 ng/m3 and from 2.22 to 36.38 ng/m3, respectively, depending on the amount of endotoxins added to the dust. Data were also obtained in a piggery. The experimental setup detailed in this paper could be used to study the significance of air contaminants in the development of pig respiratory diseases.

The effects of R 76,713 on steroidogenesis were studied in primary cultures of four different human cell types, i.e. ovarian granulosa cells, adipose stromal cells, testicular cells and adrenal cells. In human granulosa cells aromatization of [1 beta, 2 beta-3H]androstenedione (as measured by the release of tritiated water) showed a Km (Michaelis constant) of 78 nM. R 76,713 competitively inhibited aromatization with a Ki (dissociation constant of the enzyme-inhibitor complex) of 1.6 nM. In human adipose stromal cells aromatization was measured by following the conversion of androstenedione to estrone and 17 beta-estradiol. In this system a Km for aromatization of androstenedione of 10.8 nM was found. R 76,713 again showed competitive kinetics with a Ki-value of 0.14 nM. In human testicular cells the synthesis of the androgens testosterone, androstenedione and dehydroepiandrosterone was only inhibited by drug concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713, steroid concentrations were lowered to 56, 64 and 81% of the control for testosterone, androstenedione and dehydroepiandrosterone respectively. Concomitantly, a slight increase in the levels of pregnenolone (138% of the control) and progesterone (133% of the control) was seen. In human adrenal cells the synthesis of cortisol and aldosterone was slightly affected by R 76,713 also at concentrations exceeding 10(-6) M. At 10(-5) M of R 76,713 the concentrations of cortisol and aldosterone were lowered to respectively 59 and 51% of the control. At the same drug concentration the precursors 11-deoxycortisol and 11-deoxycorticosterone rose to 189 and 147% of the control. These results show that in primary cultures of human cells, R 76,713 is a very potent aromatase inhibitor with a selectivity of at least 1000-fold compared to other steps in steroidogenesis.

A method is described for the purification of canine prolactin, involving preparative isoelectrofocusing. Canine prolactin has a molecular weight of 23 000 daltons, an isoelectric point of 5.7 and exhibits a high degree of homogeneity in polyacrylamide gels stained by means of a silver method. A specific, homologous radioimmunoassay is described using the Bolton-Hunter method for preparation of the labelled ligand, with a sensitivity of 0.1 ng/tube. Basal plasma prolactin levels of 2-4 ng/ml obtained through the oestrous cycle remained fairly constant but a rise of 9 ng/ml was found at the end of dioestrus in non-pregnant bitches. Level also rose 30 days after mating to reach a peak of about 50 ng/ml near parturition and during early lactation.

Some endocrinological effects of single daily oral administration of 150 mg ketoconazole for 15 days were investigated in 4 male beagle dogs. Plasma testosterone fell markedly within 3-4 h and then progressively returned to control concentrations by 10 h after drug administration. On the other hand, plasma 17 alpha-hydroxyprogesterone, progesterone and 17 alpha, 20 alpha-dihydroxyprogesterone increased within 3-10 h before returning to basal values after 24 h. Plasma LH did not rise significantly though some high individual levels were noted. Plasma cortisol and oestradiol-17 alpha levels were not significantly modified by the treatment. These results confirm that a high therapeutic dose of ketoconazole, given orally once a day, transiently inhibits in vivo the 17-20 lyase enzyme of the testis, without modifying basal cortisol and oestradiol-17 beta plasma concentrations and that enzymatic inhibition still occurs after daily treatment for up to 2 weeks but remains transient and parallels the resorption profile of the drug so that normal plasma testosterone levels are observed from 10 to 24 h after drug intake. However, permanent inhibition of androgen biosynthesis might be obtained by the administration of high doses of ketoconazole given several times a day.

At high doses, ketoconazole blocks both testicular and adrenal androgen biosyntheses and partially inhibits the glucocorticoid production. To investigate the effects of this imidazole derivative on the mineralocorticoid biosynthesis, 7 male mongrel dogs received a single oral dose of 15 mg/kg of ketoconazole or placebo, in a cross-over way. From 2 to 4 h after treatment, an iv infusion of angiotensin II (10 ng/kg per min) was performed. Ketoconazole treatment significantly blunted the aldosterone and cortisol increment, whereas 18-hydroxycorticosterone, corticosterone, 11-deoxycorticosterone (DOC), progesterone, and 17 alpha-hydroxyprogesterone rose to peak concentrations, respectively 2.5-, 6-, 8-, 2.5- and 1.5-fold higher than those observed after placebo administration. Plasma 11-deoxycortisol and renin activity levels remained similar in both groups. On the other hand, 2 X 2 groups of 10 male adult rats each were fed with a normal or a sodium-depleted diet. Of the two sets of groups, one was treated ip with ketoconazole (20 mg/kg twice a day), the other with vehicle solution. In animals on either diet, ketoconazole lowered 18-hydroxycorticosterone and aldosterone concentrations. Plasma DOC rose up to 25-fold in the salt-deprived animals. Serum Na+, Cl-, corticosterone and plasma renin activity remained unaffected by the treatment. These results show that high-dose ketoconazole treatment partially inhibits the biosynthesis of aldosterone by affecting the cytochrome P-45011 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro, ketoconazole has been shown to block testicular and adrenal 17,20-lyase, which converts progestins to androgens. At higher concentrations, it also inhibits 11 beta-hydroxylase, 20,22-desmolase and 17 alpha-hydroxylase. To determine the differential hormonal effects of a 2-week ketoconazole high-dose therapy, the plasma levels of 10 major androgens, gluco- and mineralocorticoids were measured in 14 previously untreated patients with metastatic prostate cancer. Within 24 h, plasma testosterone fell from 14.6 +/- 1.4 nmol/l (mean +/- SEM) to 3.7 +/- 0.7 nmol/l. Thereafter, it decreased to about 2.5 nmol/l and remained at that level. Plasma androstenedione and dehydroepiandrosterone decreased more gradually, respectively from 3.1 +/- 0.4 nmol/l to 0.64 +/- 0.17 nmol/l and from 6.6 +/- 1.0 nmol/l to 2.82 +/- 0.55 nmol/l (on day 14). In contrast, 17 alpha-hydroxyprogesterone and progesterone rose respectively 2- and 5-fold. Plasma cortisol and aldosterone levels remained unchanged whereas 11-deoxycorticosterone and 11-deoxycortisol rose by factors of 14 and 6.7 respectively. Plasma corticosterone also increased, but to a much lesser extent (3-fold). These results demonstrate that ketoconazole high dose therapy blocks mainly the 17,20-lyase of both adrenal and testis. In addition it inhibits mitochondrial 11 beta-hydroxylase to a lesser extent. The inhibition of 20,22-desmolase also seems to be of little clinical relevance. However, since clinical or laboratory symptoms suggestive of hypo-adrenalism have been reported in a small minority of patients, replacement therapy should be considered in such cases.

The effects, of etomidate and of its fluoro analogue, R 8110, on adrenal, testicular and ovarian steroid biosynthesis were compared using cultures of guinea-pig adrenal, rat adrenal capsular, rat testicular and rat ovarian granulosa cells. At a concentration of 100 nM, etomidate inhibited the adrenal 11-hydroxylation of glucocorticoid and mineralocorticoid biosyntheses, producing a decrease in cortisol and corticosterone and an accumulation of 11-deoxycortisol and 11-deoxycorticosterone in guinea-pig adrenal and rat capsular adrenal cell suspensions, respectively. At higher concentrations (greater than 10(-6) M), etomidate also inhibited ovarian oestradiol production, testicular androgen formation and ovarian progesterone synthesis. The latter action suggests an effect on ovarian aromatase, on testicular 17 alpha/17,20-lyase activities and finally on cholesterol side-chain cleavage. The fluoro analogue of etomidate, R 8110, was ten times less potent as an inhibitor of 11-hydroxylation and affected progesterone formation only slightly in adrenal cell suspensions. Testosterone production was less affected by R 8110 than by etomidate. The increase of progestins suggests that the 17 alpha/17,20-lyase activities are the most sensitive testicular enzymatic reactions to R 8110. For inhibition of ovarian oestradiol production, R 8110 was twenty times more potent than etomidate.

The effects of high-dose ketoconazole (i.e. 400 mg every 8 h) therapy on adrenal steroidogenesis were investigated in 7 patients with advanced prostatic cancer who no longer responded to orchiectomy. An ACTH challenge was performed before and on days 14 and 28 of high-dose ketoconazole treatment. During the last 14 days, dexamethasone (0.5 mg twice daily) was administered together with ketoconazole. High-dose ketoconazole alone lowered the basal levels of the androgens by 49-66%. It almost completely inhibited their stimulation by ACTH, whereas plasma progesterone was doubled. Basal cortisol was only slightly lowered, but the response to ACTH stimulation was markedly blunted. Basal and stimulated plasma aldosterone remained unaffected. Both basal and stimulated 11-deoxycortisol, 11-deoxycorticosterone, and, to a lesser extent, corticosterone rose more markedly after ketoconazole than after placebo. The basal and stimulated plasma adrenal androgen levels were further reduced after combined ketoconazole-dexamethasone treatment, whereas plasma corticosterone, 11-deoxycortisol, and 11-deoxycorticosterone were lowered in the same way as cortisol. Aldosterone and progesterone profiles were similar to those observed under high-dose ketoconazole, but plasma 17 alpha-hydroxyprogesterone increased more markedly than after high-dose ketoconazole alone. These results demonstrate that high-dose ketoconazole lowers plasma androgen levels in orchiectomized patients and partly inhibits the gluco- and mineralocorticoid syntheses, especially after ACTH-stimulation. The addition of dexamethasone does not only correct the possible consequence of the impairment of the cortisol production by high-dose ketoconazole, but it further reduces the androgen levels and lowers the plasma concentrations of most precursors, for instance 11-deoxycorticosterone, which has some physiological mineralocorticoid activity.(ABSTRACT TRUNCATED AT 250 WORDS)