Compensated Hypogonadism Identified in Males with Cluster Headache: A Prospective Case-Controlled Study.

OBJECTIVE: Androgens have been hypothesized to be involved in the pathophysiology of cluster headache due to the male predominance, but whether androgens are altered in patients with cluster headache remains unclear. METHODS: We performed a prospective, case-controlled study in adult males with cluster headache. Sera were measured for hormones including testosterone, luteinizing hormone (LH), and sex hormone-binding globulin in 60 participants with episodic cluster headache (during a bout and in remission), 60 participants with chronic cluster headache, and 60 age- and sex-matched healthy controls. Free testosterone (fT) was calculated according to the Vermeulen equation. Shared genetic risk variants were assessed between cluster headache and testosterone concentrations. RESULTS: The mean fT/LH ratio was reduced by 35% (95% confidence interval [CI]: 21%-47%, p < 0.0001) in patients with chronic cluster headache and by 24% (95% CI: 9%-37%, p = 0.004) in patients with episodic cluster headache compared to controls after adjusting for age, sleep duration, and use of acute medication. Androgen concentrations did not differ between bouts and remissions. Furthermore, a shared genetic risk allele, rs112572874 (located in the intron of the microtubule associated protein tau (MAPT) gene on chromosome 17), between fT and cluster headache was identified. INTERPRETATION: Our results demonstrate that the male endocrine system is altered in patients with cluster headache to a state of compensated hypogonadism, and this is not an epiphenomenon associated with sleep or the use of acute medication. Together with the identified shared genetic risk allele, this may suggest a pathophysiological link between cluster headache and fT. ANN NEUROL 2024;95:1149-1161.

Mechanisms of GTN-induced migraine: Role of NOS isoforms, sGC and peroxynitrite in a migraine relevant mouse model.

BACKGROUND: Migraine research has highlighted the pivotal role of nitric oxide (NO) in migraine pathophysiology. Nitric oxide donors such as glyceryl trinitrate (GTN) induce migraine attacks in humans, whereas spontaneous migraine attacks can be aborted by inhibiting NO production. The present study aimed to investigate how GTN triggers migraine through its three nitric oxide synthase (NOS) isoforms (neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)) via a suspected feed-forward phenomenon. METHODS: Migraine-relevant hypersensitivity was induced by repeated injection of GTN in an in vivo mouse model. Cutaneous tactile sensitivity was assessed using von Frey filaments. Signaling pathways involved in this model were dissected using non-selective and selective NOS inhibitors, knockout mice lacking eNOS or nNOS and their wild-type control mice. Also, we tested a soluble guanylate cyclase inhibitor and a peroxynitrite decomposition catalyst (Ntotal = 312). RESULTS: Non-selective NOS inhibition blocked GTN-induced hypersensitivity. This response was partially associated with iNOS, and potentially nNOS and eNOS conjointly. Furthermore, we found that the GTN response was largely dependent on the generation of peroxynitrite and partly soluble guanylate cyclase. CONCLUSIONS: Migraine-relevant hypersensitivity induced by GTN is mediated by a possible feed-forward phenomenon of NO driven mainly by iNOS but with contributions from other isoforms. The involvement of peroxynitrite adds to the notion that oxidative stress reactions are also involved.

VPAC1 and VPAC2 receptors mediate tactile hindpaw hypersensitivity and carotid artery dilatation induced by PACAP38 in a migraine relevant mouse model.

BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide pivotal in migraine pathophysiology and is considered a promising new migraine drug target. Although intravenous PACAP triggers migraine attacks and a recent phase II trial with a PACAP-inhibiting antibody showed efficacy in migraine prevention, targeting the PACAP receptor PAC1 alone has been unsuccessful. The present study investigated the role of three PACAP receptors (PAC1, VPAC1 and VPAC2) in inducing migraine-relevant hypersensitivity in mice. METHODS: Hindpaw hypersensitivity was induced by repeated PACAP38 injections. Tactile sensitivity responses were quantified using von Frey filaments in three knockout (KO) mouse strains, each lacking one of the PACAP-receptors (Ntotal = 160). Additionally, ex vivo wire myography was used to assess vasoactivity of the carotid artery, and gene expression of PACAP receptors was examined by qPCR. RESULTS: PACAP38 induced hypersensitivity in WT controls (p < 0.01) that was diminished in VPAC1 and VPAC2 KO mice (p < 0.05). In contrast, PAC1 KO mice showed similar responses to WT controls (p > 0.05). Myograph experiments supported these findings showing diminished vasoactivity in VPAC1 and VPAC2 KO mice. We found no upregulation of the non-modified PACAP receptors in KO mice. CONCLUSIONS: This study assessed all three PACAP receptors in a migraine mouse model and suggests a significant role of VPAC receptors in migraine pathophysiology. The lack of hypersensitivity reduction in PAC1 KO mice suggests the involvement of other PACAP receptors or compensatory mechanisms. The results indicate that targeting only individual PACAP receptors may not be an effective migraine treatment.

The PACAP pathway is independent of CGRP in mouse models of migraine: possible new drug target?

Calcitonin gene-related peptide (CGRP)-antagonizing drugs represent a major advance in migraine treatment. However, up to 50% of patients do not benefit from monoclonal antibodies against CGRP or its receptor. Here, we test the hypothesis that a closely related peptide, pituitary adenylate cyclase-activating peptide (PACAP-38), works independently of CGRP and thus might represent a new, alternative drug target. To understand differences in CGRP- and PACAP-mediated migraine pain, we used mouse models of provoked migraine-like pain based on multiple stimulations and subsequent measurement of tactile sensitivity response with von Frey filaments. Genetically modified mice lacking either functional CGRP receptors (Ramp1 knockout) or TRPA1 channels (Trpa1 knockout) were used together with CGRP-targeting antibodies and chemical inhibitors in wild-type mice (ntotal = 299). Ex vivo myograph studies were used to measure dilatory responses to CGRP and PACAP-38 in mouse carotid arteries. PACAP-38 provoked significant hypersensitivity and dilated the carotid arteries independently of CGRP. In contrast, glyceryl trinitrate-induced hypersensitivity is dependent on CGRP. Contrary to previous results with the migraine-inducing substances glyceryl trinitrate, cilostazol and levcromakalim, PACAP-38-induced hypersensitivity worked only partially through inhibition of ATP-sensitive potassium channels. Using multiple migraine-relevant models, these findings establish the PACAP-38 pathway as distinct from other migraine provoking pathways such as CGRP and glyceryl trinitrate. PACAP antagonism may therefore be a novel therapeutic target of particular interest in patients unresponsive to CGRP-antagonizing drugs.

Comprehensive Evaluation of Blood Plasma and Serum Sample Preparations for HRMS-Based Chemical Exposomics: Overlaps and Specificities.

Sample preparation of biological samples can have a substantial impact on the coverage of small molecules detectable using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). This initial step is particularly critical for the detection of externally derived chemicals and their metabolites (internal chemical exposome) generally present at trace levels. Hence, our objective was to investigate how blood sample preparation methods affect the detection of low-abundant chemicals and to propose alternative methods to improve the coverage of the internal chemical exposome. We performed a comprehensive evaluation of 12 sample preparation methods (SPM) using phospholipid and protein removal plates (PLR), solid phase extraction plates (SPE), supported liquid extraction cartridge (SLE), and conventionally used protein precipitation (PPT). We implemented new quantitative and qualitative criteria for nontargeted analyses (detection frequency, recoveries, repeatability, matrix effect, low-level spiking significance, method detection limits, throughput, and ease of use) to amply characterize these SPM in a step-by-step-type approach. As a final step, PPT and one PLR plate were applied to cohort plasma and serum samples injected in triplicate to monitor batch repeatability, and annotation was performed on the related data sets to compare the respective impacts of these SPM. We demonstrate that sample preparation significantly affects both the range of observable compounds and the level at which they can be observed (only 43%-54% of total features are overlapping between the two SPM). We propose to use PPT and PLR on the same samples by implementing a simple analytical workflow as their complementarity would allow the broadening of the visible chemical space.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models.

BACKGROUND: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. METHODS: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. RESULTS: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. CONCLUSION: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

PACAP signaling is not involved in GTN- and levcromakalim-induced hypersensitivity in mouse models of migraine.

BACKGROUND: Calcitonin gene-related peptide (CGRP) antagonizing drugs represents the most important advance in migraine therapy for decades. However, these new drugs are only effective in 50-60% of patients. Recent studies have shown that the pituitary adenylate cyclase-activating peptide (PACAP38) pathway is independent from the CGRP signaling pathway. Here, we investigate PACAP38 signaling pathways in relation to glyceryl trinitrate (GTN), levcromakalim and sumatriptan. METHODS: In vivo mouse models of PACAP38-, GTN-, and levcromakalim-induced migraine were applied using tactile sensitivity to von Frey filaments as measuring readout. Signaling pathways involved in the three models were dissected using PACAP-inhibiting antibodies (mAbs) and sumatriptan. RESULTS: We showed that PACAP mAbs block PACAP38 induced hypersensitivity, but not via signaling pathways involved in GTN and levcromakalim. Also, sumatriptan has no effect on PACAP38-induced hypersensitivity relevant to migraine. This is the first study testing the effect of a PACAP-inhibiting drug on GTN- and levcromakalim-induced hypersensitivity. CONCLUSIONS: Based on the findings in our mouse model of migraine using migraine-inducing compounds and anti-migraine drugs, we suggest that PACAP acts via a distinct pathway. Using PACAP38 antagonism may be a novel therapeutic target of interest in a subgroup of migraine patients who do not respond to existing therapies.

Characterization of erenumab and rimegepant on calcitonin gene-related peptide induced responses in Xenopus Laevis oocytes expressing the calcitonin gene-related peptide receptor and the amylin-1 receptor.

BACKGROUND: The clinical use of calcitonin gene-related peptide receptor (CGRP-R) antagonists and monoclonal antibodies against CGRP and CGRP-R has offered new treatment possibilities for migraine patients. CGRP activates both the CGRP-R and structurally related amylin 1 receptor (AMY1-R). The relative effect of erenumab and the small-molecule CGRP-R antagonist, rimegepant, towards the CGRP-R and AMY-R needs to be further characterized. METHODS: The effect of CGRP and two CGRP-R antagonists were examined in Xenopus laevis oocytes expressing human CGRP-R, human AMY1-R and their subunits. RESULTS: CGRP administered to receptor expressing oocytes induced a concentration-dependent increase in current with the order of potency CGRP-R> > AMY1-R > calcitonin receptor (CTR). There was no effect on single components of the CGRP-R; calcitonin receptor-like receptor and receptor activity-modifying protein 1. Amylin was only effective on AMY1-R and CTR. Inhibition potencies (pIC50 values) for erenumab on CGRP induced currents were 10.86 and 9.35 for CGRP-R and AMY1-R, respectively. Rimegepant inhibited CGRP induced currents with pIC50 values of 11.30 and 9.91 for CGRP-R and AMY1-R, respectively. CONCLUSION: Our results demonstrate that erenumab and rimegepant are potent antagonists of CGRP-R and AMY1-R with 32- and 25-times preference for the CGRP-R over the AMY1-R, respectively. It is discussed if this difference in affinity between the two receptors is the likely reason why constipation is a common and serious adverse effect during CGRP-R antagonism but less so with CGRP binding antibodies.

Microbial genome analysis: the COG approach.

For the past 20 years, the Clusters of Orthologous Genes (COG) database had been a popular tool for microbial genome annotation and comparative genomics. Initially created for the purpose of evolutionary classification of protein families, the COG have been used, apart from straightforward functional annotation of sequenced genomes, for such tasks as (i) unification of genome annotation in groups of related organisms; (ii) identification of missing and/or undetected genes in complete microbial genomes; (iii) analysis of genomic neighborhoods, in many cases allowing prediction of novel functional systems; (iv) analysis of metabolic pathways and prediction of alternative forms of enzymes; (v) comparison of organisms by COG functional categories; and (vi) prioritization of targets for structural and functional characterization. Here we review the principles of the COG approach and discuss its key advantages and drawbacks in microbial genome analysis.

No additive effect of combining sumatriptan and olcegepant in the GTN mouse model of migraine.

INTRODUCTION: Despite recent advances in migraine treatment there is a need for therapies with higher clinical efficacy and/or fewer side effects. Triptans (5-HT1B/1D/1F agonists) are essential in the present treatment regime and gepants (CGRP-receptor antagonists) are recognized as effective in acute migraine treatment. Triptans and gepants have different mechanisms of action and here we tested the hypothesis that a combination of these drugs (sumatriptan and olcegepant) would result in an additive effect. METHODS: Using the validated glyceryl trinitrate mouse model of migraine, we initially tested dose-response relationships of sumatriptan (0.1, 0.3, and 0.6 mg/kg IP) and olcegepant (0.25, 0.50, and 1.0 mg/kg IP) to find suitable high and low doses. Subsequently, we performed a combination study of the two drugs with a low and a high dose. All experiments were vehicle (placebo) controlled and blinded. RESULTS: Sumatriptan significantly reduced glyceryl trinitrate-induced allodynia (F(4,54) = 13.51, p < 0.0001) at all doses. Olcegepant also reduced glyceryl trinitrate-induced allodynia (F(4,53) = 16.11, p < 0.0001) with the two higher doses being significantly effective. Combining 0.50 mg/kg olcegepant with 0.1 or 0.6 mg/kg sumatriptan did not have any improved effect compared to either drug alone (p > 0.50 on all days) in our mouse model. CONCLUSION: Combining olcegepant and sumatriptan did not have an additive effect compared to single-drug treatment in this study. Triptan-gepant combinations will therefore most likely not improve migraine treatment. Nevertheless, further studies are necessary, and combinations should also be examined in patients with migraine.

CGRP-dependent signalling pathways involved in mouse models of GTN- cilostazol- and levcromakalim-induced migraine.

BACKGROUND: Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. METHODS: In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. RESULTS: Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. CONCLUSION: The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine mechanisms.

Functional gene networks reveal distinct mechanisms segregating in migraine families.

Migraine is the most common neurological disorder worldwide and it has been shown to have complex polygenic origins with a heritability of estimated 40-70%. Both common and rare genetic variants are believed to underlie the pathophysiology of the prevalent types of migraine, migraine with typical aura and migraine without aura. However, only common variants have been identified so far. Here we identify for the first time a gene module with rare mutations through a systems genetics approach integrating RNA sequencing data from brain and vascular tissues likely to be involved in migraine pathology in combination with whole genome sequencing of 117 migraine families. We found a gene module in the visual cortex, based on single nuclei RNA sequencing data, that had increased rare mutations in the migraine families and replicated this in a second independent cohort of 1930 patients. This module was mainly expressed by interneurons, pyramidal CA1, and pyramidal SS cells, and pathway analysis showed association with hormonal signalling (thyrotropin-releasing hormone receptor and oxytocin receptor signalling pathways), Alzheimer's disease pathway, serotonin receptor pathway and general heterotrimeric G-protein signalling pathways. Our results demonstrate that rare functional gene variants are strongly implicated in the pathophysiology of migraine. Furthermore, we anticipate that the results can be used to explain the critical mechanisms behind migraine and potentially improving the treatment regime for migraine patients.

Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism.

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

No central action of CGRP antagonising drugs in the GTN mouse model of migraine.

INTRODUCTION: Clinically, calcitonin gene-related peptide antagonising drugs are recognized as effective in migraine treatment, but their site of action is debated. Only a small fraction of these compounds pass the blood-brain barrier and accesses the central nervous system. Regardless, it has been argued that the central nervous system is the site of action. Here, we test this hypothesis by bypassing the blood-brain barrier through intracerebroventricular injection of calcitonin gene-related peptide antagonising drugs. METHODS: We used the glyceryl trinitrate (GTN) mouse model, which is well validated by its response to specific migraine drugs. The calcitonin gene-related peptide receptor antagonist olcegepant and the calcitonin gene-related peptide monoclonal antibody ALD405 were administered either intraperitoneally or intracerebroventricularly. The outcome measure was cutaneous mechanical allodynia. RESULTS: Mice given olcegepant intraperitoneally + GTN on day 1 had a mean 50% withdrawal threshold of 1.2 g in contrast to mice receiving placebo + GTN, which had a threshold of 0.3 g (p < 0.001). Similarly, in the ALD405 + GTN group, mice had thresholds of 1.2 g versus 0.2 g in the placebo + GTN group (p < 0.001). However, both drugs were ineffective when delivered intracerebroventricularly, as control and active groups had identical mechanical sensitivity thresholds, 0.2 g versus 0.1 g and 0.1 g versus 0.1 g for olcegepant and ALD405, respectively (p > 0.99 in both cases). DISCUSSION: The site of action of olcegepant and of the monoclonal antibody ALD405 is outside the blood-brain barrier in this mouse model of migraine. It is likely that these results can be generalised to all gepants and all antibodies and that the results are relevant for human migraine.

ATP sensitive potassium (KATP) channel inhibition: A promising new drug target for migraine.

BACKGROUND: Recently, the adenosine triphosphate (ATP) sensitive potassium channel opener levcromakalim was shown to induce migraine attacks with a far higher incidence than any previous provoking agent such as calcitonin gene-related peptide. Here, we show efficacy of ATP sensitive potassium channel inhibitors in two validated rodent models of migraine. METHODS: In female spontaneous trigeminal allodynic rats, the sensitivity of the frontal region of the head was tested by an electronic von Frey filament device. In mice, cutaneous hypersensitivity was induced by repeated glyceryl trinitrate or levcromakalim injections over nine days, as measured with von Frey filaments in the hindpaw. Release of calcitonin gene-related peptide from dura mater and trigeminal ganglion was studied ex vivo. RESULTS: The ATP sensitive potassium channel inhibitor glibenclamide attenuated the spontaneous cephalic hypersensitivity in spontaneous trigeminal allodynic rats and glyceryl trinitrate-induced hypersensitivity of the hindpaw in mice. It also inhibited CGRP release from dura mater and the trigeminal ganglion isolated from spontaneous trigeminal allodynic rats. The hypersensitivity was also diminished by the structurally different ATP sensitive potassium channel inhibitor gliquidone. Mice injected with the ATP sensitive potassium channel opener levcromakalim developed a progressive hypersensitivity that was completely blocked by glibenclamide, confirming target engagement. CONCLUSION: The results suggest that ATP sensitive potassium channel inhibitors could be novel and highly effective drugs in the treatment of migraine.

Targeting CGRP via receptor antagonism and antibody neutralisation in two distinct rodent models of migraine-like pain.

INTRODUCTION: Rodent disease models can play an indispensable role in drug development. Confirming that translationally-relevant disease mechanisms are engaged in such models is a crucial facet of this process. Accordingly, we have validated the role of calcitonin gene-related peptide signaling in a mouse model of glyceryl trinitrate-provoked migraine-like pain and a spontaneous rat model of migraine-like pain by assessing their pharmacological responsiveness to the small molecule calcitonin gene-related peptide receptor antagonist olcegepant, and the humanised monoclonal calcitonin gene-related peptide antibody ALD405. METHODS: Cutaneous sensitivity to hind paw, and periorbital mechanical stimulation were used as surrogate markers of activation of relevant pain pathways in each respective model. Separate experiments were performed to identify the time-course of treatment response to olcegepant (1 mg/kg i.p.) and ALD405 (10 mg/kg i.p.). RESULTS: Olcegepant and ALD405 significantly alleviated cutaneous mechanical hypersensitivity in both models compared with corresponding control treatments (saline and IgG control antibody respectively). As expected, the duration of anti-nociceptive action obtained with ALD405 was considerably longer than that associated with olcegepant. Surprisingly, in the spontaneous rat model the onset of action of ALD405 occurred within just 4 hours after administration. DISCUSSION: The current data clearly show that calcitonin gene-related peptide-mediated signaling is critically involved in the manifestation of cutaneous hypersensitivity in distinct rodent models of migraine-like pain and emphasise their translational relevance. Moreover, the unexpected rapidity of onset observed for ALD405 supports i) a probable site of action outside the blood-brain barrier, and ii) a potential clinical utility of specific monoclonal calcitonin gene-related peptide antibodies in the abortive treatment of migraine.

The Ramazzini Institute 13-week pilot study glyphosate-based herbicides administered at human-equivalent dose to Sprague Dawley rats: effects on development and endocrine system.

BACKGROUND: Glyphosate-based herbicides (GBHs) are broad-spectrum herbicides that act on the shikimate pathway in bacteria, fungi, and plants. The possible effects of GBHs on human health are the subject of an intense public debate for both its potential carcinogenic and non-carcinogenic effects, including potential effects on the endocrine system The present pilot study examine whether exposure to GBHs at the dose of glyphosate considered to be "safe" (the US Acceptable Daily Intake - ADI - of 1.75 mg/kg bw/day), starting from in utero life, affect the development and endocrine system across different life stages in Sprague Dawley (SD) rats. METHODS: Glyphosate alone and Roundup Bioflow, a commercial brand of GBHs, were administered in drinking water at 1.75 mg/kg bw/day to F0 dams starting from the gestational day (GD) 6 (in utero) up to postnatal day (PND) 120. After weaning, offspring were randomly distributed in two cohorts: 8 M + 8F/group animals belonging to the 6-week cohort were sacrificed after puberty at PND 73 ± 2; 10 M + 10F/group animals belonging to the 13-week cohort were sacrificed at adulthood at PND 125 ± 2. Effects of glyphosate or Roundup exposure were assessed on developmental landmarks and sexual characteristics of pups. RESULTS: In pups, anogenital distance (AGD) at PND 4 was statistically significantly increased both in Roundup-treated males and females and in glyphosate-treated males. Age at first estrous (FE) was significantly delayed in the Roundup-exposed group and serum testosterone concentration significantly increased in Roundup-treated female offspring from the 13-week cohort compared to control animals. A statistically significant increase in plasma TSH concentration was observed in glyphosate-treated males compared with control animals as well as a statistically significant decrease in DHT and increase in BDNF in Roundup-treated males. Hormonal status imbalances were more pronounced in Roundup-treated rats after prolonged exposure. CONCLUSIONS: The present pilot study demonstrate that GBHs exposure, from prenatal period to adulthood, induced endocrine effects and altered reproductive developmental parameters in male and female SD rats. In particular, it was associated with androgen-like effects, including a statistically significant increase of AGDs in both males and females, delay of FE and increased testosterone in female.

Prokaryotic Virus Orthologous Groups (pVOGs): a resource for comparative genomics and protein family annotation.

Viruses are the most abundant and diverse biological entities on earth, and while most of this diversity remains completely unexplored, advances in genome sequencing have provided unprecedented glimpses into the virosphere. The Prokaryotic Virus Orthologous Groups (pVOGs, formerly called Phage Orthologous Groups, POGs) resource has aided in this task over the past decade by using automated methods to keep pace with the rapid increase in genomic data. The uses of pVOGs include functional annotation of viral proteins, identification of genes and viruses in uncharacterized DNA samples, phylogenetic analysis, large-scale comparative genomics projects, and more. The pVOGs database represents a comprehensive set of orthologous gene families shared across multiple complete genomes of viruses that infect bacterial or archaeal hosts (viruses of eukaryotes will be added at a future date). The pVOGs are constructed within the Clusters of Orthologous Groups (COGs) framework that is widely used for orthology identification in prokaryotes. Since the previous release of the POGs, the size has tripled to nearly 3000 genomes and 300 000 proteins, and the number of conserved orthologous groups doubled to 9518. User-friendly webpages are available, including multiple sequence alignments and HMM profiles for each VOG. These changes provide major improvements to the pVOGs database, at a time of rapid advances in virus genomics. The pVOGs database is hosted jointly at the University of Iowa at http://dmk-brain.ecn.uiowa.edu/pVOGs and the NCBI at ftp://ftp.ncbi.nlm.nih.gov/pub/kristensen/pVOGs/home.html.

ATGC database and ATGC-COGs: an updated resource for micro- and macro-evolutionary studies of prokaryotic genomes and protein family annotation.

The Alignable Tight Genomic Clusters (ATGCs) database is a collection of closely related bacterial and archaeal genomes that provides several tools to aid research into evolutionary processes in the microbial world. Each ATGC is a taxonomy-independent cluster of 2 or more completely sequenced genomes that meet the objective criteria of a high degree of local gene order (synteny) and a small number of synonymous substitutions in the protein-coding genes. As such, each ATGC is suited for analysis of microevolutionary variations within a cohesive group of organisms (e.g. species), whereas the entire collection of ATGCs is useful for macroevolutionary studies. The ATGC database includes many forms of pre-computed data, in particular ATGC-COGs (Clusters of Orthologous Genes), multiple sequence alignments, a set of 'index' orthologs representing the most well-conserved members of each ATGC-COG, the phylogenetic tree of the organisms within each ATGC, etc. Although the ATGC database contains several million proteins from thousands of genomes organized into hundreds of clusters (roughly a 4-fold increase since the last version of the ATGC database), it is now built with completely automated methods and will be regularly updated following new releases of the NCBI RefSeq database. The ATGC database is hosted jointly at the University of Iowa at dmk-brain.ecn.uiowa.edu/ATGC/ and the NCBI at ftp.ncbi.nlm.nih.gov/pub/kristensen/ATGC/atgc_home.html.

Evolutionary switches between two serine codon sets are driven by selection.

Serine is the only amino acid that is encoded by two disjoint codon sets so that a tandem substitution of two nucleotides is required to switch between the two sets. Previously published evidence suggests that, for the most evolutionarily conserved serines, the codon set switch occurs by simultaneous substitution of two nucleotides. Here we report a genome-wide reconstruction of the evolution of serine codons in triplets of closely related species from diverse prokaryotes and eukaryotes. The results indicate that the great majority of codon set switches proceed by two consecutive nucleotide substitutions, via a threonine or cysteine intermediate, and are driven by selection. These findings imply a strong pressure of purifying selection in protein evolution, which in the case of serine codon set switches occurs via an initial deleterious substitution quickly followed by a second, compensatory substitution. The result is frequent reversal of amino acid replacements and, at short evolutionary distances, pervasive homoplasy.