Efficacy of m-Health for the detection of adverse events following immunization - The stimulated telephone assisted rapid safety surveillance (STARSS) randomised control trial.

INTRODUCTION: Passive surveillance is recommended globally for the detection of adverse events following immunisation (AEFI) but this has significant challenges. Use of Mobile health for vaccine safety surveillance enables a consumer-centred approach to reporting. The Stimulated Telephone Assisted Rapid Safety Surveillance (STARSS) a randomised control trial (RCT) sought to evaluate the efficacy and acceptability of SMS for AEFI surveillance. METHODS: Multi-centre RCT, participants were adult vaccinees or parents of children receiving any vaccine at a trial site. At enrolment randomisation occurred to one of two SMS groups or a control group. Prompts on days 2, 7 and 14 post-immunisation, were sent to the SMS group, to ascertain if a medical event following immunisation (MEFI) had occurred. No SMS's were sent to the control participants. Those in the SMS who notified an MEFI were pre-randomised to complete a computer assisted telephone interview or a web based report to determine if an AEFI had occurred whilst an AEFI in the controls was determined by a search for passive reports. The primary outcome was the AEFI detection rate in the SMS group compared to controls. RESULTS: We enrolled 6,338 participants, who were equally distributed across groups and who received 11,675 vaccines. The SMS group (4,225) received 12,675 surveillance prompts with 9.8% being non-compliant and not responding. In those that responded 90% indicated that no MEFI had been experienced and 184 had a verified AEFI. 6 control subjects had a reported AEFI. The AEFI detection rate was 13 fold greater in the SMS group when compared with controls (4.3 vs 0.3%). CONCLUSION: We have demonstrated that the STARSS methodology improves AEFI detection. Our findings should inform the wider use of SMS-based surveillance which is particularly relevant since establishing robust and novel pharmacovigilance systems is critical to monitoring novel vaccines which includes potential COVID vaccines.

Consumer acceptability and validity of m-Health for the detection of adverse events following immunization - The Stimulated Telephone Assisted Rapid Safety Surveillance (STARSS) randomised control trial.

INTRODUCTION: Monitoring for adverse events following immunisation (AEFI) is critical for vaccine pharmacovigilance. Given the global and expanding availability of mobile phones their utility for consumer-based vaccine safety surveillance is of interest but little is known about consumer acceptability. This study nested within the Stimulated Telephone Assisted Rapid Safety Surveillance (STARSS) randomised control trial sought to evaluate the acceptability of SMS for AEFI surveillance. METHODS: The primary STARSS study was a multi-centre RCT evaluating the efficacy of repeated SMS prompts for AEFI surveillance with participants being adult vaccinees or parents of children receiving any vaccine. This nested study enrolled primary RCT participants who completed a detailed computer assisted telephone interview to determine their attitudes towards SMS-based surveillance and ascertain their knowledge and attitudes toward vaccine safety, efficacy, data privacy and use of electronic health records. Attitudes to surveillance and related behaviour were used as measures of acceptability. RESULTS: 20% (1200/6555) of the participants were enrolled and 1139 completed the full-length questionnaire. 96% indicated that SMS-based surveillance after immunisation to check the safety of the vaccine "should be done" but 62% of all respondents said it should be done but consent should be sought first. Neither vaccine safety attitudes nor attitudes toward privacy were associated with opposition to SMS-based surveillance. In terms of SMS related behaviour demographic rather than attitudinal factors were associated with non-compliance. CONCLUSION: Overall, the attitude towards SMS-based surveillance was very favourable. Experiencing the SMS surveillance has the effect of reducing opposition to an SMS surveillance system, and at the same time increasing the likelihood of a preference for prior consent. Detection of a vaccine safety signal could be impeded in particular demographic groups who are non-compliant and we should undertake further research to understand why these groups are non-compliant and how this can be improved.

Voriconazole-natural latex dressings for treating infected Candida spp. skin ulcers.

Aim: This work aimed to develop a membrane based on voriconazole (VCZ)-loaded natural rubber latex (NRL) for treating infected ulcers with Candida spp. and study their interaction, drug release, antifungal activity against Candida parapsilosis and biological characterization. Materials & methods: VCZ-loaded NRL membrane was produced by casting method. Results: Infrared spectrum showed that the incorporation of VCZ into the NRL membrane maintained its characteristics. Its mechanical properties were considered suitable for dermal application. The VCZ was able to release from NRL membrane, maintaining its antifungal activity against C. parapsilosis, besides did not present hemolytic effects. Conclusion: The VCZ-NRL membrane showed good results in mechanical, antifungal and biological assays, representing an interesting alternative to treatment of infected wound with Candida spp.

Mechanism of Rate Acceleration of Radical C-C Bond Formation Reaction by a Radical SAM GTP 3',8-Cyclase.

While the number of characterized radical S-adenosyl-l-methionine (SAM) enzymes is increasing, the roles of these enzymes in radical catalysis remain largely ambiguous. In radical SAM enzymes, the slow radical initiation step kinetically masks the subsequent steps, making it impossible to study the kinetics of radical chemistry. Due to this kinetic masking, it is unknown whether the subsequent radical reactions require rate acceleration by the enzyme active site. Here, we report the first evidence that a radical SAM enzyme MoaA accelerates the radical-mediated C-C bond formation. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterizations, we found that MoaA catalyzes a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from (4'R)-5'-deoxyadenosine (5'-dA) to transiently generate 5'-deoxyadenos-4'-yl radical (5'-dA-C4'•) that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics and determined the rate of the on-pathway 3',8-cyclization step as 2.7 ± 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization by MoaA is accelerated by 6-9 orders of magnitude. Further experimental and theoretical characterizations suggested that the rate acceleration is achieved mainly by constraining the triphosphate and guanine base positions while leaving the ribose flexible, and a transition state stabilization through H-bond and electrostatic interactions with the positively charged R17 residue. This is the first evidence for rate acceleration of radical reactions by a radical SAM enzyme and provides insights into the mechanism by which radical SAM enzymes accelerate radical chemistry.

Paradigm Shift for Radical S-Adenosyl-l-methionine Reactions: The Organometallic Intermediate Ω Is Central to Catalysis.

Radical S-adenosyl-l-methionine (SAM) enzymes comprise a vast superfamily catalyzing diverse reactions essential to all life through homolytic SAM cleavage to liberate the highly reactive 5'-deoxyadenosyl radical (5'-dAdo·). Our recent observation of a catalytically competent organometallic intermediate Ω that forms during reaction of the radical SAM (RS) enzyme pyruvate formate-lyase activating-enzyme (PFL-AE) was therefore quite surprising, and led to the question of its broad relevance in the superfamily. We now show that Ω in PFL-AE forms as an intermediate under a variety of mixing order conditions, suggesting it is central to catalysis in this enzyme. We further demonstrate that Ω forms in a suite of RS enzymes chosen to span the totality of superfamily reaction types, implicating Ω as essential in catalysis across the RS superfamily. Finally, EPR and electron nuclear double resonance spectroscopy establish that Ω involves an Fe-C5' bond between 5'-dAdo· and the [4Fe-4S] cluster. An analogous organometallic bond is found in the well-known adenosylcobalamin (coenzyme B12) cofactor used to initiate radical reactions via a 5'-dAdo· intermediate. Liberation of a reactive 5'-dAdo· intermediate via homolytic metal-carbon bond cleavage thus appears to be similar for Ω and coenzyme B12. However, coenzyme B12 is involved in enzymes catalyzing only a small number (∼12) of distinct reactions, whereas the RS superfamily has more than 100 000 distinct sequences and over 80 reaction types characterized to date. The appearance of Ω across the RS superfamily therefore dramatically enlarges the sphere of bio-organometallic chemistry in Nature.

Cross-Sectional Analysis of Commonly Prescribed Medications in Military Aviation.

BACKGROUND: Medication use by naval aviators, either prescription or over-the-counter, is not always relayed to the flight surgeon, resulting in unsafe flying environments. Many medications have debilitating effects that prohibit their use during aviation. Education and availability of resources on approved medications for flight status personnel is lacking. METHODS: A retrospective search of the Department of Defense Composite Health Care System (DoD CHCS) was conducted from five geographic locations. Basic epidemiological information was obtained to determine the most common medications (N = 70) prescribed to active-duty flight status personnel. Analysis determined their medication category, flight status designation, and a generalized location comparison. A similar control medication list for nonflight status personnel was generated from one location. RESULTS: Analysis found that many medications prescribed to aviators are not approved for use in aviation and are similar to those of nonflight status personnel. There were 8 of the top 15 (53%) and 40 of the 70 (57%) most commonly prescribed medications of flight status personnel which were not approved. Similarly, 49% of total prescriptions (N = 15,652) were not approved. Little difference was found in medications among nonflight and flight status personnel, as 11 of the top 15 prescription medications (73%) were identical. DISCUSSION: This research demonstrates the need for education regarding certain medications and their prohibited use during flight. Results will provide the Flight Surgeon common medications prescribed to aviators to aid them in providing a safer flying environment.Ropp LG, Haight SP, Prudhomme MB, Ropp EL. Cross-sectional analysis of commonly prescribed medications in military aviation. Aerosp Med Hum Perform. 2017; 88(12):1129-1133.

¹9F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR.

Naturally occurring RNA lacks fluorine-19 ((19)F), thus, their specifically fluorinated counterparts are particularly well suited to noninvasively monitoring the dynamic conformational properties and ligand-binding interactions of the RNA. For nuclear magnetic resonance (NMR) spectroscopy, (19)F-NMR of fluorine-substituted RNA provides an attractive, site-specific probe for structure determination in solution. Advantages of (19)F include high NMR sensitivity (83% of (1)H), high natural abundance (100%), and the extreme sensitivity of (19)F to the chemical environment leading to a large range of chemical shifts. The preparation of base-substituted 2-fluoropurine and 5-fluoropyrimidine 5'-triphosphates (2F-ATP/5F-CTP/5F-UTP) can be carried out using efficient enzymatic synthesis methods. Both pyrimidine analogs, 5-fluorouridine and 5-fluorocytidine, as well as, 2-fluoroadenosine are readily incorporated into RNA transcribed in vitro using T7 RNA polymerase.

Disrupted seasonal biology impacts health, food security and ecosystems.

The rhythm of life on earth is shaped by seasonal changes in the environment. Plants and animals show profound annual cycles in physiology, health, morphology, behaviour and demography in response to environmental cues. Seasonal biology impacts ecosystems and agriculture, with consequences for humans and biodiversity. Human populations show robust annual rhythms in health and well-being, and the birth month can have lasting effects that persist throughout life. This review emphasizes the need for a better understanding of seasonal biology against the backdrop of its rapidly progressing disruption through climate change, human lifestyles and other anthropogenic impact. Climate change is modifying annual rhythms to which numerous organisms have adapted, with potential consequences for industries relating to health, ecosystems and food security. Disconcertingly, human lifestyles under artificial conditions of eternal summer provide the most extreme example for disconnect from natural seasons, making humans vulnerable to increased morbidity and mortality. In this review, we introduce scenarios of seasonal disruption, highlight key aspects of seasonal biology and summarize from biomedical, anthropological, veterinary, agricultural and environmental perspectives the recent evidence for seasonal desynchronization between environmental factors and internal rhythms. Because annual rhythms are pervasive across biological systems, they provide a common framework for trans-disciplinary research.

Aeromedical Risk Assessment of Pharmaceuticals Using Evidence-Based Medicine.

BACKGROUND: Using concepts from evidence-based medicine, systems theory, and risk assessment, a standardized model was developed to accept or reject medications for use in flight. The model calculates the risk scores of medications, which can then be compared to an organization's acceptable risk tolerance. METHODS: Risk scores for each medication were established by summing the products of incidence rates and severity scores for all published side effects. The incidence of each side effect was obtained in an evidence-based manner and each assigned a severity multiplier. Using statistical analysis of the calculated risk scores of approved medications, an acceptance control chart was generated. RESULTS: Range of calculated risk scores of historically approved medications was 10-9140. Six Sigma Acceptance Control Line was calculated at 1.5 SDs above the mean and was 9822. Risk score range of medications generally felt unsafe was 27,010-41,294. Risk score range of medications under consideration for approval was 986-6863. DISCUSSION: This novel approach to medication approval is the first in aerospace medicine to attempt to combine evidence-based medicine, risk analysis, and control charts to standardize and streamline the medication approval process within an organization. The model was validated by testing against medications generally accepted to be unsafe for use in flight. These medications fell several deviations above the control line. Other medications not yet authorized fall well below the acceptance line and could be considered for approval.

Photoperiodic variation in CD45-positive cells and cell proliferation in the mediobasal hypothalamus of the Soay sheep.

The Earth's solar orbit induces annual climatic changes challenging to survival. Many animals have evolved to cope with seasonal variability through compensatory annual changes in their physiology and behavior, which involve innate long-term timing and photoperiodic synchronization to anticipate the environmental seasonal cycles. Here we considered the potential involvement of cyclical histogenesis in seasonal timing mechanisms in the sheep. Adult Soay rams were established in three distinctive seasonal states by controlled photoperiod exposure. A first group, representing the condition in late spring (long-photoperiod [LP] group), was taken indoors in May and exposed to 4 wks of 16 h light/day (LP). A second group was exposed to 20 wks of LP to establish a late-summer/long-day refractory condition (LPR group). A third group of animals was brought indoors in August and exposed to 4 wks of LP followed by 4 wks of 8 h light/day (short photoperiod [SP]) to establish an autumn-like condition (SP group). At the end of these regimes, we injected 5-bromo-2-deoxyuridine (BrdU), and animals were killed 24 h or 4 wks later. When BrdU was administered 24 h before death, more BrdU-immunopositive cells were detected in the hilus of the hippocampus in LP compared with SP animals, indicative of a higher proliferation rate. When BrdU was administered 4 wks before death, more BrdU-positive cells were detected in the hippocampus under LP, compared with SP, indicating increased cell survival. These mitotic cells were occasionally seen to adopt a neuronal phenotype in the hippocampus, but not in the hypothalamus. Approximately 10% of BrdU-positive cells in the basal hypothalamus coexpressed the pan-leukocytic marker CD45, and showed morphological features and regional distribution consistent with ameboid microglia. Increased numbers of these cells were detected in the region of the median eminence and tuberoinfundibular sulcus of animals kept in SP compared with LP or LPR. These data suggest that neuroimmune mechanisms may be involved in photoperiod-dependent seasonal remodeling of the adult brain.

Circannual variation in thyroid hormone deiodinases in a short-day breeder.

At temperate latitudes, many mammals and birds show internally timed, long-term changes in seasonal physiology, synchronised to the seasons by changing day length (photoperiod). Photoperiodic control of thyroid hormone levels in the hypothalamus dictates the timing. This is effected through reciprocal regulation of thyroid hormone deiodinase gene expression. The local synthesis of type 2 deiodinase (Dio2) promotes triiodothyronine (T3) production and summer biology, whereas type 3 deiodinase (Dio3) promotes T3 degradation and winter biology. In the present study, we investigated the extent to which the hypothalamic expression of Dio2 and Dio3 is circannually regulated in the Soay sheep, a short-day breeding mammal. Male sheep were exposed to a long photoperiod (LP; 16 : 24 h light/dark cycle) or a short photoperiod (SP; 8 : 24 h light/dark cycle), for up to 28 weeks to establish four different endocrine states: (i) LP animals in a spring/summer-like state of reproductive arrest; (ii) LP refractory (LPR) animals showing spontaneous reproductive reactivation; (iii) SP animals showing autumn/winter-like reproductive activation; and (iv) SP refractory (SPR) animals showing spontaneous reproductive arrest. A complex pattern of hypothalamic Dio2 and Dio3 expression was observed, revealing distinctive photoperiod-driven and internally timed effects for both genes. The patterns of expression differed both spatially and temporally, with phases of peak Dio2 expression in the median eminence and tuberoinfundibular sulcus, as well as in the paraventricular zone (PVZ) (maximal under LP), whereas Dio3 expression was always confined to the PVZ (maximal under SP). These effects likely reflect the distinct roles of these enzymes in the localised control of hypothalamic T3 levels. The spontaneous decline in Dio2 and spontaneous increase in Dio3 in LPR animals occurred with a corresponding decline in thyroid-stimulating hormone ß expression in the neighbouring pars tuberalis (PT), although this relationship did not hold for the corresponding Dio2 increase/Dio3 decrease seen in SPR animals. We conclude that internally timed and spatially regulated changes in Dio2 and Dio3 expression may drive the cycling between breeding and nonbreeding states in long-lived seasonal species, and may be either PT-dependent or PT-independent at different phases of the circannual cycle.

An active-site guanine participates in glmS ribozyme catalysis in its protonated state.

Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.

Evidence for RGS4 modulation of melatonin and thyrotrophin signalling pathways in the pars tuberalis.

In mammals, the pineal hormone melatonin is secreted nocturnally and acts in the pars tuberalis (PT) of the anterior pituitary to control seasonal neuroendocrine function. Melatonin signals through the type 1 Gi-protein coupled melatonin receptor (MT1), inhibiting adenylate cyclase (AC) activity and thereby reducing intracellular concentrations of the second messenger, cAMP. Because melatonin action ceases by the end of the night, this allows a daily rise in cAMP levels, which plays a key part in the photoperiodic response mechanism in the PT. In addition, melatonin receptor desensitisation and sensitisation of AC by melatonin itself appear to fine-tune this process. Opposing the actions of melatonin, thyroid-stimulating hormone (TSH), produced by PT cells, signals through its cognate Gs-protein coupled receptor (TSH-R), leading to increased cAMP production. This effect may contribute to increased TSH production by the PT during spring and summer, and is of considerable interest because TSH plays a pivotal role in seasonal neuroendocrine function. Because cAMP stands at the crossroads between melatonin and TSH signalling pathways, any protein modulating cAMP production has the potential to impact on photoperiodic readout. In the present study, we show that the regulator of G-protein signalling RGS4 is a melatonin-responsive gene, whose expression in the PT increases some 2.5-fold after melatonin treatment. Correspondingly, RGS4 expression is acutely sensitive to changing day length. In sheep acclimated to short days (SP, 8 h light/day), RGS4 expression increases sharply following dark onset, peaking in the middle of the night before declining to basal levels by dawn. Extending the day length to 16 h (LP) by an acute 8-h delay in lights off causes a corresponding delay in the evening rise of RGS4 expression, and the return to basal levels is delayed some 4 h into the next morning. To test the hypothesis that RGS4 expression modulates interactions between melatonin- and TSH-dependent cAMP signalling pathways, we used transient transfections of MT1, TSH-R and RGS4 in COS7 cells along with a cAMP-response element luciferase reporter (CRE-luc). RGS4 attenuated MT1-mediated inhibition of TSH-stimulated CRE-luc activation. We propose that RGS4 contributes to photoperiodic sensitivity in the morning induction of cAMP-dependent gene expression in the PT.

The pH dependence of hairpin ribozyme catalysis reflects ionization of an active site adenine.

Understanding how self-cleaving ribozymes mediate catalysis is crucial in light of compelling evidence that human and bacterial gene expression can be regulated through RNA self-cleavage. The hairpin ribozyme catalyzes reversible phosphodiester bond cleavage through a mechanism that does not require divalent metal cations. Previous structural and biochemical evidence implicated the amidine group of an active site adenosine, A38, in a pH-dependent step in catalysis. We developed a way to determine microscopic pK(a) values in active ribozymes based on the pH-dependent fluorescence of 8-azaadenosine (8azaA). We compared the microscopic pK(a) for ionization of 8azaA at position 38 with the apparent pK(a) for the self-cleavage reaction in a fully functional hairpin ribozyme with a unique 8azaA at position 38. Microscopic and apparent pK(a) values were virtually the same, evidence that A38 protonation accounts for the decrease in catalytic activity with decreasing pH. These results implicate the neutral unprotonated form of A38 in a transition state that involves formation of the 5'-oxygen-phosphorus bond.

Enzymatic de novo pyrimidine nucleotide synthesis.

The use of stable isotope labeling has revolutionized NMR studies of nucleic acids, and there is a need for methods of incorporation of specific isotope labels to facilitate specific NMR experiments and applications. Enzymatic synthesis offers an efficient and flexible means to synthesize nucleoside triphosphates from a variety of commercially available specifically labeled precursors, permitting isotope labeling of RNAs prepared by in vitro transcription. Here, we recapitulate de novo pyrimidine biosynthesis in vitro, using recombinantly expressed enzymes to perform efficient single-pot syntheses of UTP and CTP that bear a variety of stable isotope labeling patterns. Filtered NMR experiments on (13)C, (15)N, (2)H-labeled HIV-2 TAR RNA demonstrate the utility and value of this approach. This flexible enzymatic synthesis will make implementing detailed and informative RNA stable isotope labeling schemes substantially more cost-effective and efficient, providing advanced tools for the study of structure and dynamics of RNA molecules.

Influence of organic carbon loading, sediment associated metal oxide content and sediment grain size distributions upon Cryptosporidium parvum removal during riverbank filtration operations, Sonoma County, CA.

This study assessed the efficacy for removing Cryptosporidium parvum oocysts of poorly sorted, Fe- and Al-rich, subsurface sediments collected from 0.9 to 4.9 and 1.7-13.9 m below land surface at an operating riverbank filtration (RBF) site (Russian River, Sonoma County, CA). Both formaldehyde-killed oocysts and oocyst-sized (3 microm) microspheres were employed in sediment-packed flow-through and static columns. The degree of surface coverage of metal oxides on sediment grain surfaces correlated strongly with the degrees of oocyst and microsphere removals. In contrast, average grain size (D(50)) was not a good indicator of either microsphere or oocyst removal, suggesting that the primary mechanism of immobilization within these sediments is sorptive filtration rather than physical straining. A low specific UV absorbance (SUVA) for organic matter isolated from the Russian River, suggested that the modest concentration of the SUVA component (0.8 mg L(-1)) of the 2.2 mg L(-1) dissolved organic carbon (DOC) is relatively unreactive. Nevertheless, an amendment of 2.2 mg L(-1) of isolated river DOC to column sediments resulted in up to a 35.7% decrease in sorption of oocysts and (or) oocyst-sized microspheres. Amendments (3.2 microM) of the anionic surfactant, sodium dodecyl benzene sulfonate (SDBS) also caused substantive decreases (up to 31.9 times) in colloid filtration. Although the grain-surface metal oxides were found to have a high colloid-removal capacity, our study suggested that any major changes within the watershed that would result in long-term alterations in either the quantity and (or) the character of the river's DOC could alter the effectiveness of pathogen removal during RBF operations.

Mammalian circannual pacemakers.

Circannual clocks drive rhythms in reproduction and many other seasonal characteristics but the underlying control of these long-term oscillators remains a mystery. Now, we propose that circannual timing involves mechanisms that are integral to the ontogenetic life-history programme where annual transitions are generated by cell birth, death and tissue regeneration throughout the life cycle--the histogenesis hypothesis. The intrinsic cycle is then timed by cues from the environment. The concept is that in specific sites in the brain, pituitary and peripheral tissues, residual populations of progenitor cells (adult stem cells) synchronously initiate a phase of cell division to begin a cycle. The progeny cells then proliferate, migrate and differentiate, providing the substrate that drives physiological change over long time-spans (e.g. summer/winter); cell death may be required to trigger the next cycle. We have begun to characterise such a tissue-based timer in our Soay sheep model focusing on the pars tuberalis (PT) of the pituitary gland and the sub-ventricular zone of the mediobasal hypothalamus (MBH) as potential circannual pacemakers. The PT is of special interest because it is a melatonin-responsive tissue containing undifferentiated cells, strategically located at the gateway between the brain and pituitary gland. The PT also governs long-photoperiod activation of thyroid hormone dependant processes in the MBH required for neurogenesis. In sheep, exposure to long photoperiod markedly activates BrDU-labelled cell proliferation in the PT and MBH, and acts to entrain the circannual reproductive cycle. Variation in expression and co-ordination of multiple tissue timers may explain species differences in circannual rhythmicity. This paper is dedicated to the memory of Ebo Gwinner.

A case of adult acute idiopathic scrotal edema.

BACKGROUND: A 40-year-old man presented to a hospital emergency department with acute-onset, bilateral, painless swelling of the scrotum, which had developed progressively over the previous 12 h. He was physically well with no remarkable medical history, and was not taking any medication. He had no history of allergies or trauma (including insect bites to the genitalia), no urinary symptoms, and was not sexually active. INVESTIGATIONS: Physical examination, assessment of erythrocyte sedimentation rate, white blood cell count, renal function panel, urine microscopic analysis, urine cultures, transillumination, and scrotal ultrasonography. DIAGNOSIS: Acute idiopathic scrotal edema. MANAGEMENT: The patient received expectant management and treatment with an NSAID to relieve his inflammation. He was discharged from hospital and his symptoms resolved within 72 h of onset of treatment.

Direct measurement of the ionization state of an essential guanine in the hairpin ribozyme.

Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme in order to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8, and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.

Egr1 involvement in evening gene regulation by melatonin.

Seasonal photoperiodic responses in mammals depend on the pineal hormone melatonin. The pars tuberalis (PT) region of the anterior pituitary has emerged as a principal melatonin target tissue, controlling endocrine responses. Rising melatonin levels acutely influence the expression of a small cluster of genes either positively (exemplified by cryptochrome-1, cry1) or negatively (exemplified by the type 1 melatonin receptor, mt1). The purpose of this study was to characterize the pathways through which these evening actions of melatonin are mediated. In vitro experiments showed that cAMP signaling in the PT directly influences mt1 but not cry1 expression. Analysis of nuclear extracts from sheep PT tissue collected 90 min after melatonin or saline control injections highlighted the response element for the immediate early gene egr1 (EGR1-RE) as a candidate for acute melatonin-dependent transcriptional regulation. We identified putative EGR1-RE's in the proximal promoter regions of the ovine cry1 and mt1 genes, and confirmed their functionality in luciferase reporter assays. Egr1 expression is suppressed by melatonin in PT cell cultures, and is rhythmic in the ovine PT with a nadir in the early night. We propose that melatonin-dependent effects on EGR1-RE's contribute to evening gene expression profiles in this pituitary melatonin target tissue.