Detection of prions from spiked and free-ranging carnivore feces.

Chronic wasting disease (CWD) is a highly contagious, fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting wild and captive cervids. Although experimental feeding studies have demonstrated prions in feces of crows (Corvus brachyrhynchos), coyotes (Canis latrans), and cougars (Puma concolor), the role of scavengers and predators in CWD epidemiology remains poorly understood. Here we applied the real-time quaking-induced conversion (RT-QuIC) assay to detect PrPCWD in feces from cervid consumers, to advance surveillance approaches, which could be used to improve disease research and adaptive management of CWD. We assessed recovery and detection of PrPCWD by experimental spiking of PrPCWD into carnivore feces from 9 species sourced from CWD-free populations or captive facilities. We then applied this technique to detect PrPCWD from feces of predators and scavengers in free-ranging populations. Our results demonstrate that spiked PrPCWD is detectable from feces of free-ranging mammalian and avian carnivores using RT-QuIC. Results show that PrPCWD acquired in natural settings is detectable in feces from free-ranging carnivores, and that PrPCWD rates of detection in carnivore feces reflect relative prevalence estimates observed in the corresponding cervid populations. This study adapts an important diagnostic tool for CWD, allowing investigation of the epidemiology of CWD at the community-level.

First Observation of Cyclotron Radiation from MeV-Scale e

We present an apparatus for detection of cyclotron radiation yielding a frequency-based ß^{±} kinetic energy determination in the 5 keV to 2.1 MeV range, characteristic of nuclear ß decays. The cyclotron frequency of the radiating ß particles in a magnetic field is used to determine the ß energy precisely. Our work establishes the foundation to apply the cyclotron radiation emission spectroscopy (CRES) technique, developed by the Project 8 Collaboration, far beyond the 18-keV tritium endpoint region. We report initial measurements of ß^{-}'s from ^{6}He and ß^{+}'s from ^{19}Ne decays to demonstrate the broadband response of our detection system and assess potential systematic uncertainties for ß spectroscopy over the full (MeV) energy range. To our knowledge, this is the first direct observation of cyclotron radiation from individual highly relativistic ß's in a waveguide. This work establishes the application of CRES to a variety of nuclei, opening its reach to searches for new physics beyond the TeV scale via precision ß-decay measurements.

Ticks harbor and excrete chronic wasting disease prions.

Chronic wasting disease (CWD) is a fatal neurodegenerative disease caused by infectious prions (PrPCWD) affecting cervids. Circulating PrPCWD in blood may pose a risk for indirect transmission by way of hematophagous ectoparasites acting as mechanical vectors. Cervids can carry high tick infestations and exhibit allogrooming, a common tick defense strategy between conspecifics. Ingestion of ticks during allogrooming may expose naïve animals to CWD, if ticks harbor PrPCWD. This study investigates whether ticks can harbor transmission-relevant quantities of PrPCWD by combining experimental tick feeding trials and evaluation of ticks from free-ranging white-tailed deer (Odocoileus virginianus). Using the real-time quaking-induced conversion (RT-QuIC) assay, we show that black-legged ticks (Ixodes scapularis) fed PrPCWD-spiked blood using artificial membranes ingest and excrete PrPCWD. Combining results of RT-QuIC and protein misfolding cyclic amplification, we detected seeding activity from 6 of 15 (40%) pooled tick samples collected from wild CWD-infected white-tailed deer. Seeding activities in ticks were analogous to 10-1000 ng of CWD-positive retropharyngeal lymph node collected from deer upon which they were feeding. Estimates revealed a median infectious dose range of 0.3-42.4 per tick, suggesting that ticks can take up transmission-relevant amounts of PrPCWD and may pose a CWD risk to cervids.

ß-Nuclear-Recoil Correlation from

We report the first precise measurement of a ß-recoil correlation from a radioactive noble gas (^{6}He) confined via a magneto-optical trap. The measurement is motivated by the search for exotic tensor-type contributions to the charged weak current. Interpreted as tensor currents with right-handed neutrinos, the measurements yield |C_{T}/C_{A}|^{2}≤0.022 (90% confidence limit, C.L.). On the other hand, for left-handed neutrinos the limits are 0.007

SARS-CoV-2 in first trimester pregnancy: a cohort study.

STUDY QUESTION: Does maternal infection with severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) in first trimester pregnancy have an impact on the fetal development as measured by nuchal translucency thickness and pregnancy loss? SUMMARY ANSWER: Nuchal translucency thickness at the first trimester scan was not significantly different in pregnant women with versus without SARS-CoV-2 infection in early pregnancy and there was no significantly increased risk of pregnancy loss in women with SARS-CoV-2 infection in the first trimester. WHAT IS KNOWN ALREADY: Pregnant women are more vulnerable to viral infections. Previous coronavirus epidemics have been associated with increased maternal morbidity, mortality and adverse obstetric outcomes. Currently, no evidence exists regarding possible effects of SARS-CoV-2 in first trimester pregnancies. STUDY DESIGN, SIZE, DURATION: Cohort study of 1019 women with a double test taken between 17 February and 23 April 2020, as a part of the combined first trimester risk assessment, and 36 women with a first trimester pregnancy loss between 14 April and 21 May 2020, prior to the double test. The study period was during the first SARS-CoV-2 epidemic wave in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cohort 1 included pregnant women with a double test taken within the study period. The excess serum from each double test was analyzed for SARS-CoV-2 antibodies. Results were correlated to the nuchal translucency thickness and the number of pregnancy losses before or at the time of the first trimester scan. Cohort 2 included women with a pregnancy loss before the gestational age for double test sample. Serum from a blood test taken the day the pregnancy loss was identified was analyzed for SARS-CoV-2 antibodies. The study was conducted at a public university hospital serving ∼12% of pregnant women and births in Denmark. All participants in the study provided written informed consent. MAIN RESULTS AND THE ROLE OF CHANCE: Eighteen (1.8%) women had SARS-CoV-2 antibodies in the serum from the double test suggestive of SARS-CoV-2 infection in early pregnancy. There was no significant difference in nuchal translucency thickness for women testing positive for previous SARS-CoV-2 infection (n = 16) versus negative (n = 966) (P = 0.62). There was no significantly increased risk of pregnancy loss for women with antibodies (n = 1) (OR 3.4, 0.08-24.3 95% CI, P = 0.27). None of the women had been hospitalized due to SARS-CoV-2 infection. None of the women with pregnancy loss prior to the double test (Cohort 2) had SARS-CoV-2 antibodies. LIMITATIONS, REASONS FOR CAUTION: These results may only apply to similar populations and to patients who do not require hospitalization due to SARS-CoV-2 infection. A limitation of the study is that only 1.8% of the study population had SARS-CoV-2 antibodies suggestive of previous infection. WIDER IMPLICATION OF THE FINDINGS: Maternal SARS-CoV-2 infection had no effect on the nuchal translucency thickness and there was no significantly increased risk of pregnancy loss for women with SARS-CoV-2 infection in first trimester pregnancy. Evidence concerning COVID-19 in pregnancy is still limited. These data indicate that infection with SARS-CoV-2 in not hospitalized women does not pose a significant threat in first trimester pregnancies. Follow-up studies are needed to establish any risk to a fetus exposed to maternal SARS-CoV-2 infection. STUDY FUNDING/COMPETING INTEREST(S): Prof. H.S.N. and colleagues received a grant from the Danish Ministry of Research and Education for research of COVID-19 among pregnant women. The Danish government was not involved in the study design, data collection, analysis, interpretation of data, writing of the report or decision to submit the paper for publication. A.I., J.O.-L., J.B.-R., D.M.S., J.E.-F. and E.R.H. received funding from a Novo Nordisk Foundation (NNF) Young Investigator Grant (NNF15OC0016662) and a Danish National Science Foundation Center Grant (6110-00344B). A.I. received a Novo Scholarship. J.O.-L. is funded by an NNF Pregraduate Fellowship (NNF19OC0058982). D.W. is funded by the NNF (NNF18SA0034956, NNF14CC0001, NNF17OC0027594). A.M.K. is funded by a grant from the Rigshospitalet's research fund. H.S.N. has received speaker's fees from Ferring Pharmaceuticals, Merck Denmark A/S and Ibsa Nordic (outside the submitted work). N.l.C.F. has received a grant from Gedeon Richter (outside the submitted work). A.M.K. has received speaker's fee from Merck (outside the submitted work). The other authors did not report any potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Assessment and validation of a screening questionnaire for the diagnosis of pediatric bladder and bowel dysfunction.

INTRODUCTION: Pediatric bladder and bowel dysfunction (BBD) is a common problem in children. However, the current ability to diagnosis and quantify pediatric BBD is limited as only a few validated instruments exist. In addition, the current questionnaires are limited by their lack of psychometric processing and methods of validation. To address these issues, the authors developed a new questionnaire to objectively diagnose pediatric BBD symptoms. This study aimed to evaluate the performance of this newly devised objective instrument in diagnosing and quantifying the symptomatology of BBD in children. MATERIALS AND METHODS: An 18-item, 5-point questionnaire was developed using both a literature review and expert opinions. The total questionnaire score could range from 0 to 72. Questions were subgrouped into six symptom categories: (1) nocturnal enuresis, (2) lower urinary tract symptoms, (3) urinary holding, (4) infrequent urination, (5) bowel symptoms, and (6) daytime urinary incontinence. The questionnaire also assessed the degree of bother associated with the symptoms. Patients were divided into cases and controls, and these two groups were compared. DISCUSSION/RESULTS: A total of 1265 new patients (758 cases and 507 controls) completed the new BBD questionnaire. The mean age of the whole study cohort was 9.5 years (range, 3-19 years). The total mean questionnaire score was significantly higher at 23 (3-58) in the cases, compared with 8 (0-35) in the controls (p < 0.001) (Summary Figure). Reliability analysis of the 18-item instrument showed a Cronbach's alpha reliability coefficient of 0.80 for the scale. CONCLUSIONS: This new instrument provides a valid and reliable method for diagnosis of pediatric BBD and classification of patients into subcategories of BBD based on their specific symptoms.

The Iowa Voiding Improvement Partnership experience: Early observations with a collaborative pediatric uro-psychologic clinic.

INTRODUCTION: Bladder and bowel dysfunction (BBD) are common problems in children presenting for pediatric urology referral. Psychiatric issues may be present in these children, making their treatment difficult. In 2013, the University of Iowa Voiding Improvement Partnership (VIP) Clinic was established for the treatment of these patients. STUDY OBJECTIVE: This study sought to evaluate early experience with this specialized clinic, to determine the pre-existing urologic and psychologic conditions seen in these clinic patients, and to evaluate the clinical outcomes after VIP treatment. STUDY DESIGN: A retrospective, Institutional Review Board-approved review of all patients seen in the VIP Clinic was performed. The following were evaluated: patient demographics, underlying urologic and psychologic diagnosis, and treatment decisions. All patients were asked to complete the University of Iowa Pediatric Bladder and Bowel Dysfunction questionnaire at each visit. Questionnaire scores from the patients' first and most recent clinic visits were compared. RESULTS: To date, 66 patients have been evaluated at the VIP Clinic, accounting for 112 clinic visits. The mean age of the VIP patients was 8.5 years (range, 4-16) and 59% of the patients were female. Pre-existing urological conditions and psychological conditions are shown in the Summary Table. A large number (62%) of patients required further psychological evaluation, secondary to concern for an undiagnosed psychiatric issue. In addition, the clinic had improved patients' BBD symptoms over time. When first evaluated in clinic, patients had an average Iowa BBD Questionnaire score of 31 (range, 47-13), which improved to an average score of 25 (range, 47-7) (P = 0.03). In addition, 23% of the patients improved to where they could be discharged from uro-psychologic care. CONCLUSIONS: It was feasible to establish the present multidisciplinary uro-psychology clinic. Such a clinic may unearth undiagnosed psychological issues, and improve bowel and bladder dysfunction in these difficult-to-treat patients.

Inter-rater reliability of distal ureteral diameter ratio compared to grade of VUR.

INTRODUCTION AND OBJECTIVE: Management of vesicoureteral reflux (VUR) remains controversial, and reflux grade constitutes an important prognostic factor. Recent work has suggested that distal ureteral diameter ratio (UDR) is a predictive factor relative to clinical outcome independent of grade. Previous studies have noted significant inter-rater variability with grading of VUR. The present study compared inter-rater reliability of reflux grade and UDR in children with primary VUR. STUDY DESIGN: Four pediatric urologists independently reviewed, in a blinded fashion, voiding cystourethrograms. For each renal unit, grade was assigned according to the standardized international scale. The UDR was calculated by dividing the largest ureteral diameter within the false pelvis by the distance between L1-L3 vertebral bodies. Correlation within each rater was determined using Pearson's correlation coefficient. Reliability of VUR grade and UDR were calculated using two-way ANOVA model inter-rater agreement. RESULTS: Four independent raters reliably measured VUR grade (ICC = 0.87, 95% CI = 0.78-0.93) and UDR (ICC = 0.95, 95% CI = 0.92-0.97). While UDR and grade were equally reliable measures, UDR had a tighter confidence interval. For each rater, grade and UDR were well correlated (r = 0.73-0.84; P < 0.0001). For higher-grade reflux, grade was more variable than UDR (Summary Figure). Using empirical thresholds, the increased variability of grade compared with UDR may lead to significant differences in clinical decision-making among physicians (P = 0.022). DISCUSSION: Known discordance with grading reflux emphasizes the need for a more objective VUR measurement, as clinicians and parents often opt for clinical intervention based on both clinical course and the likelihood of spontaneous resolution. While ICC for UDR and grade were not significantly different, the confidence intervals for grade were wider due to greater variability among grade measurements. This suggests that using UDR measurements may lead to more accurate characterization of VUR and ultimately more consistent clinical decision-making across providers. CONCLUSIONS: Ureteral diameter ratio has good inter-rater reliability among pediatric urologists, with less clinically relevant variability than VUR grade. Ureteral diameter ratio is a more objective and reliable measure than grade, and may be more useful in clinical decision-making.

Utility of the distal ureteral diameter on VCUG for grading VUR.

INTRODUCTION: For children with VUR the grade of vesicoureteral reflux (VUR) remains one of the most predictive factors relative to outcome. However, the subjective nature of the currently accepted international reflux grading system (IRGS) leads to inter-observer variation. The potential of a direct measurement of the distal ureter on the voiding cystourethrogram (VCUG) normalized to the L1-L3 vertebral body distance (ureteral diameter ratio - UDR) to augment the ability of IRGS to predict the ultimate clinical outcome has previously been reported in a group of 79 children. OBJECTIVE: The goal of this current review was to expand the previous review and analysis in order to assess the predictive ability of the UDR with respect to earlier clinical outcome and to compare this ability to the grade of VUR while controlling for other variables. STUDY DESIGN: This retrospective review of the VCUG of 157 children with primary VUR included 124 girls and 33 boys with a mean age of 2.7 years (7 days-13.5 years). In addition to the UDR, other variables that were analyzed included: age, gender, VUR grade, laterality, history of febrile urinary tract infection (UTI) or multiple UTIs prior to diagnosis, and bladder-bowel dysfunction (BBD). Cox regression analysis was utilized and a generalized logit model for 2-year outcome was also fitted to compare the effect of UDR and VUR grade using Wald Chi-squared analysis. RESULTS: The 2-year outcome after the VCUG was defined as: persistent VUR (47%), spontaneous VUR resolution (15%), or operative intervention (38%). Reasons for operative intervention included: breakthrough UTI (1/3), decreased relative renal function associated with renal scarring (1/3), and failure to resolve, along with parental preference (1/3). Increasing UDR was significantly associated with increased grade and a decreased chance of spontaneous resolution. It was unlikely for a child with Grade 4, 3, or 2 VUR to have spontaneous resolution if their UDR was above 0.25, 0.3, or 0.35, respectively. In addition, higher grades of VUR, older age, and bilateral VUR were significantly associated with failure to spontaneously resolve VUR. As seen in Figure, children with grades 2 and 3 VUR less than 2 years of age had a better chance of spontaneous resolution with a larger UDR than children 2 years of age or older. When adjusting for age, grade, laterality and multiple UTIs as covariates, each unit increase of UDR of 0.1 was significantly associated with either persistent VUR (OR = 1.73, 95% CI = 1.02-2.95, P = 0.043) or the need for surgical intervention (OR = 2.40, 95% CI = 1.39-4.17, P = 0.002) compared to spontaneous resolution. When testing the effect of UDR and grade of reflux in the same model, UDR was noted to have a larger effect on predicting failure to spontaneously resolve VUR than grade (Wald Chi-Squared 13.6; P = 0.001 vs 3.62; P = 0.46, respectively). DISCUSSION: The UDR is a readily available objective measurement on the VCUG that has demonstrated ability to enhance the International Reflux Grading System. Limitations of the current review include operative intervention in 12% of the children for failure to improve or resolve VUR. This surgical intervention inhibits determination of spontaneous resolution rates. The findings in this study reflect those in a series of children from a single institution and, therefore, may be impacted by clinical practice bias and geographic variations. Subsequent multi-institutional studies could further define the potential of UDR as either an independent or additive predictive factor for grading VUR that will further permit individualized patient management. CONCLUSION: In this single institution series, UDR was highly correlated with VUR grade; however, UDR proved more predictive of spontaneous resolution, persistence, or operative intervention than grade.

Acute focal bacterial nephritis due to methicillin-resistant Staphylococcus aureus in an immunocompetent adult.

Acute focal bacterial nephritis (AFBN) is a rare, acute focal infection of the renal parenchyma without liquefaction. The pathogenesis is thought to be due to hematogenous infection or ascending infection from the lower urinary tract. Escherichia coli has been the major pathogen isolated in prior cases, but other Gram-negative enteric pathogens and Staphylococcus aureus have been reported as well. It is well described in children and adults with diabetes and organ transplantation, but has not been previously reported in healthy adults. We report a case of an immunocompetent adult female who presented with a methicillin-resistant Staphylococcus aureus bacteremia after a skin and soft tissue infection that resulted in AFBN.

Constitutive µ-opioid receptor activity leads to long-term endogenous analgesia and dependence.

Opioid receptor antagonists increase hyperalgesia in humans and animals, which indicates that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced µ-opioid receptor (MOR) constitutive activity (MOR(CA)) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the posthyperalgesia state with MOR inverse agonists reinstated central pain sensitization and precipitated hallmarks of opioid withdrawal (including adenosine 3',5'-monophosphate overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1. Thus, MOR(CA) initiates both analgesic signaling and a compensatory opponent process that generates endogenous opioid dependence. Tonic MOR(CA) suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Inactivation of Pde8b enhances memory, motor performance, and protects against age-induced motor coordination decay.

Phosphodiesterases (PDEs) are critical regulatory enzymes in cyclic nucleotide signaling. PDEs have diverse expression patterns within the central nervous system (CNS), show differing affinities for cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and regulate a vast array of behaviors. Here, we investigated the expression profile of the PDE8 gene family members Pde8a and Pde8b in the mouse brain. We find that Pde8a expression is largely absent in the CNS; by contrast, Pde8b is expressed in select regions of the hippocampus, ventral striatum, and cerebellum. Behavioral analysis of mice with Pde8b gene inactivation (PDE8B KO) demonstrate an enhancement in contextual fear, spatial memory, performance in an appetitive instrumental conditioning task, motor-coordination, and have an attenuation of age-induced motor coordination decline. In addition to improvements observed in select behaviors, we find basal anxiety levels to be increased in PDE8B KO mice. These findings indicate that selective antagonism of PDE8B may be an attractive target for enhancement of cognitive and motor functions; however, possible alterations in affective state will need to be weighed against potential therapeutic value.

In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens.

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.

Direct measurements of ²²Na(p,γ)²³Mg resonances and consequences for ²²Na production in classical novae.

The radionuclide 22Na is a potential astronomical observable that is expected to be produced in classical novae in quantities that depend on the thermonuclear rate of the 22Na(p,γ)23Mg reaction. We have measured the strengths of low-energy 22Na(p,γ)23Mg resonances directly and absolutely using a radioactive 22Na target. We find the strengths of resonances at Ep=213, 288, 454, and 610 keV to be higher than previous measurements by factors of 2.4-3.2, and we exclude important contributions to the rate from proposed resonances at Ep=198, 209, and 232 keV. The 22Na abundances expected in the ejecta of classical novae are reduced by a factor of ≈2.

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function.

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.

Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo.

Although accumulating evidence indicates that cAMP response element-binding protein (CREB) phosphorylation mediates not only synaptic plasticity but also survival of certain neurons, it remains uncertain whether CREB phosphorylation induced after metabolic insult leads to CRE-mediated gene transcription and is involved in cell survival or not. In the present study, we clarified that (1) CREB phosphorylation and ischemic tolerance induced after preconditioning ischemia in the hippocampal neurons was abolished by MK801 administration in gerbil global ischemia model, (2) CREB phosphorylation induced after exposure to glutamate in cultured neurons was inhibited by removal of extracellular calcium, by MK801 and by an inhibitor of calcium-calmodulin-dependent protein kinase (CaMK) II and IV, (3) inhibitor of CaMK II-IV or CRE-decoy oligonucleotide suppressed upregulation of BCL-2 expression and accelerated neuronal damage after exposure to glutamate, and (4) CREB phosphorylation induced in the hippocampal neurons after ischemia and in cultured neurons after exposure to glutamate was followed by CRE-mediated gene transcription in transgenic mice with a CRE-LacZ reporter. Our results suggest that CREB phosphorylation in neurons after ischemia and exposure to glutamate is induced by NMDA receptor-gated calcium influx and subsequent activation of CaMK II-IV and that CREB phosphorylation after metabolic stress might show a neuroprotective response through CRE-mediated gene induction.

The CRE/CREB pathway is transiently expressed in thalamic circuit development and contributes to refinement of retinogeniculate axons.

The development of precise connections in the mammalian brain proceeds through refinement of initially diffuse patterns, a process that occurs largely within critical developmental windows. To elucidate the molecular pathways that orchestrate these early periods of circuit remodeling, we have examined the role of a calcium- and cAMP-regulated transcriptional pathway. We show that there is a window of CRE/CREB-mediated gene expression in the developing thalamus, which precedes neocortical expression. In the LGN, this wave of gene expression occurs prior to visual experience, but requires retinal function. Mutant mice with reduced CREB expression show loss of refinement of retinogeniculate projections. These results suggest an important role of the CRE/CREB transcriptional pathway in the coordination of experience-independent circuit remodeling during forebrain development.

Adenylyl cyclase 3 mediates prostaglandin E(2)-induced growth inhibition in arterial smooth muscle cells.

Arterial smooth muscle cell (SMC) proliferation contributes to a number of vascular pathologies. Prostaglandin E(2) (PGE(2)), produced by the endothelium and by SMCs themselves, acts as a potent SMC growth inhibitor. The growth-inhibitory effects of PGE(2) are mediated through activation of G-protein-coupled membrane receptors, activation of adenylyl cyclases (ACs), formation of cAMP, and subsequent inhibition of mitogenic signal transduction pathways in SMCs. Of the 10 different mammalian AC isoforms known today, seven isoforms (AC2-7 and AC9) are expressed in SMCs from various species. We show that, despite the presence of several different AC isoforms, the principal AC isoform activated by PGE(2) in human arterial SMCs is a calmodulin kinase II-inhibited AC with characteristics similar to those of AC3. AC3 is expressed in isolated human arterial SMCs and in intact aorta. We further show that arterial SMCs isolated from AC3-deficient mice are resistant to PGE(2)-induced growth inhibition. In summary, AC3 is the principal AC isoform activated by PGE(2) in arterial SMCs, and AC3 mediates the growth-inhibitory effects of PGE(2). Because AC3 activity is inhibited by intracellular calcium through calmodulin kinase II, AC3 may serve as an important integrator of growth-inhibitory signals that stimulate cAMP formation and growth factors that increase intracellular calcium.