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Hydro-Quebec launched several energy savings programs in 1992. The programs, which will be finished in 2010 at a cost of around $3.9 billion, are expected to reduce electricity demand in Quebec by 9.3 trillion watt-hours by 2000 and 18 trillion watt-hours by 2010. Cost-benefit analysis reveals that the present value benefits of the programs will range from $2.097 billion to $4.493 billion over the period from 1992 to 2010, depending on the amount of electricity saved. Although beneficial to consumers and Hydro-Quebec, the programs are expected to result in heavy losses for the government.

We now know RNA can survive the harsh environment of biofluids when encapsulated in vesicles or by associating with lipoproteins or RNA binding proteins. These extracellular RNA (exRNA) play a role in intercellular signaling, serve as biomarkers of disease, and form the basis of new strategies for disease treatment. The Extracellular RNA Communication Consortium (ERCC) hosted a two-day online workshop (April 19-20, 2021) on the unique challenges of exRNA data analysis. The goal was to foster an open dialog about best practices and discuss open problems in the field, focusing initially on small exRNA sequencing data. Video recordings of workshop presentations and discussions are available (https://exRNA.org/exRNAdata2021-videos/). There were three target audiences: experimentalists who generate exRNA sequencing data, computational and data scientists who work with those groups to analyze their data, and experimental and data scientists new to the field. Here we summarize issues explored during the workshop, including progress on an effort to develop an exRNA data analysis challenge to engage the community in solving some of these open problems., Competing Interests: EV is co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Eclipse BioInnovations. EV’s interests have been reviewed and approved by the Baylor College of Medicine in accordance with its conflict of interest policies. RS is a paid consultant for Lacerta Therapeutics. Author RY was employed by the company Aginome Scientific, Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alexander, Kitchen, Tosar, Roth, Mestdagh, Max, Rozowsky, Kaczor-Urbanowicz, Chang, Balaj, Losic, Van Nostrand, LaPlante, Mateescu, White, Yu, Milosavljevic, Stolovitzky and Spengler.)

Background: Understanding the underlying factors that determine the relative abundance of plant species is critical to predict both biodiversity and ecosystem function. Biotic and abiotic factors can shape the distribution and the relative abundance of species across natural communities, greatly influencing local biodiversity., Methods: Using a combination of an observational study and a five-year plant removal experiment we: (1) documented how plant diversity and composition of montane meadow assemblages vary along a plant dominance gradient using an observational study; (2) tracked above- and belowground functional traits of co-dominant plant species Potentilla and Festuca along a plant dominance gradient in an observational study; (3) determined whether plant species diversity and composition was directly influenced by commonly occurring species Potentilla and Festuca with the use of a randomized plot design, 5-year plant removal experiment (no removal control, Potentilla removed, Festuca removed, n  = 10)., Results: We found that subordinate species diversity and compositional dissimilarity were greatest in Potentilla and Festuca co-dominated sites, where neither Potentilla nor Festuca dominated, rather than at sites where either species became dominant. Further, while above- and belowground plant functional traits varied along a dominance gradient, they did so in a way that inconsistently predicted plant species relative abundance. Also, neither variation in plant functional traits of Festuca and Potentilla nor variation in resources and conditions (such as soil nitrogen and temperature) explained our subordinate diversity patterns. Finally, neither Potentilla nor Festuca influenced subordinate diversity or composition when we directly tested for their impacts in a plant removal experiment., Discussion: Taken together, patterns of subordinate diversity and composition were likely driven by abiotic factors rather than biotic interactions. As a result, the role of abiotic factors influencing local-level species interactions can be just as important as biotic interactions themselves in structuring plant communities., Competing Interests: The authors declare there are no competing interests.

A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha 1-receptor (alpha 1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha 1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha 1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha 1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.

Unlabelled: Mediobasal hypothalamic slices of adult ovariectomized (OVX) rats treated or not with 17 beta-estradiol (E2) were superfused in buffered (pH 7.2) Locke medium containing bacitracin. A 6-min pulse of K+ (56 m M) was less effective in releasing luteinizing hormone releasing hormone (LH-RH) from mediobasal hypothalamic slices sampled from OVX rats than from OVX animals treated subcutaneously with either E2 or stilbestrol implants for 5 days; in contrast, the basal release of the neuropeptide was identical in both cases. Direct addition to the superfusion medium of 17 beta-estradiol (10(-10) to 10(-7) M) or stilbestrol (10(-8) M) potentiated the K+-induced LH-RH release from slices of OVX animals. The K+-induced LH-RH release observed after in vivo E2 implantation was not further amplified by in vitro addition of the hormone. Tamoxifen and hydroxytamoxifen, estrogen antagonists, were ineffective by themselves, but reversed the E2 facilitation of K+-evoked LH-RH release. In contrast, 17 alpha-estradiol, progesterone, or cholesterol (10(-8) or 10(-9) M) hat no effect on either basal or stimulated release of the neurohormone. Somatostatin release measured under identical conditions was not affected by castration or by in vitro addition of the steroid., In Conclusion: (1) estradiol appears selectively and specifically involved in the process coupling, nerve endings depolarization, and LH-RH release, and (2) the effect is receptor-mediated and does not appear to require nuclear translocation of the steroid or transcription processes, since it can be readily elicited upon addition of the hormone to nerve endings disconnected from their cell bodies.

Mediobasal hypothalamic (MBH) slices of adult ovariectomized (OVX) rats with or without 17 beta-estradiol (E2) pretreatment, were superfused in buffered (pH 7.2) oxygenated Locke medium containing bacitracin. Pulsatile or continuous administration of progesterone (10(-7) or 10(-8) M) produced a marked increase in luteinizing hormone-releasing hormone (LHRH) release provided the animals had received E2 prior to sacrifice. Omission of Ca++ in the medium, or addition of a Ca++ channel blocker (D-600, 10(-4) M), of a calmodulin inhibitor (trifluoperazine, 30 microM) or of a calmodulin-dependent tubulin kinase inhibitor (phenytoin, 50 microM), antagonized the stimulatory effect of progesterone. When sodium channels were blocked by tetrodotoxin (5 X 10(-7) M), the stimulatory effect of the steroid was completely abolished. The amplitude of the K+-induced LHRH release was slightly increased in the presence of progesterone (10(-7) M) but only from MBH slices of OVX-E2-treated rats. These results indicate that the secretory response of LHRH to progesterone requires priming with estradiol, is Ca++-dependent and involves mediation of calmodulin and a calmodulin-dependent kinase system.

The circadian pattern of LH release observed in ovariectomized estrogen-implanted rats was inhibited by blockade of alpha-adrenergic receptors by phenoxybenzamine; in contrast, blockade of beta-receptors by propranolol was ineffective. Prasozin, an alpha 1-antagonist, was as effective as phenoxybenzamine, whereas yohimbine, an alpha 2-antagonist, was effective only at high doses. Neither phenoxybenzamine nor prazosin were able to modify the LHRH-induced release of LH from superfused pituitaries. These results suggest an involvement of hypothalamic alpha 1-receptors in the control of circadian LH release.

Subcutaneous implantation of a silastic tubing containing crystalline estradiol in castrated female rats results in a circadian rhythm of LH release. Under such conditions, blockade of serotonin (5-HT) biosynthesis by p-chlorophenylalanine (PCPA) completely inhibits the afternoon elevation of plasma LH. Gonadotropin peaks remain inhibited as long as the concentration of the transmitter is effectively depleted. Intraperitoneal administration or intraventricular infusion of minute doses of the immediate precursor of the amine, 5-hydroxytryptophan (5-HTP), results in the reappearance of the next afternoon rise of plasma gonadotropin, whenever hypothalamic levels of 5-hydroxyindole acetic acid (5-HIAA), the main metabolite of 5-HT, are significantly increased over their value in PGPA-treated animals. The administration of methiothepin, a 5-HT receptor blocker, 9 hours of more prior to the next expected LH rise, similarly inhibits the cycle, whereas a dopamine receptor inhibitor has no effect under the same time conditions. We have concluded that serotoninergic neuron systems can have a positive, permissive effect on the transfer of neural information resulting in phasic gonadotropin release; this action of the amine is different from, but not contradictory to, the known inhibitory effect of 5-HT on the release of LHRH from the median eminence.

The effects of long-term anesthesia with methoxyflurane on plasma LH and prolactin concentrations were compared to those induced by exposure to ether. Methoxyflurane significantly decreased prolactin levels and had a slight effect of LH, whereas ether produced an important release of prolactin without affecting LH.

Luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) release was assessed in superfused slices of mediobasal hypothalamus. Release of both neurohormones by depolarizing agents (K+, 56mM ; veratridine, 50 muM) was shown to be Ca2+-dependent, according with the stimulus-secretion coupling hypothesis. Opiates (beta endorphin, 10(-7)M and D-ALA2-Met-enkephalinamide 10(-7)M) did not alter the spontaneous release of LHRH and SRIF, but inhibited significantly the K+-induced neuropeptide release. The effect was reversed by the opiate antagonist naloxone (10(-7)M), while naloxone was ineffective by itself. Vasoactive intestinal peptide (VIP 10(-9)M) significantly inhibited K+ evoked release of SRIF ; LHRH release was unaffected. The effect of VIP on SRIF release was dose-dependent ; secretin, a partial VIP agonist, was also active at higher doses. The data suggest that : 1) opiates, acting through specific opiate receptors located on LHRH and SRIF neurons, modulate the release of the neurohormones ; 2) the inhibitory effect of opiates could be due to an inhibition of calcium influx through voltage-dependent calcium channels ; 3) this interaction may account for the stimulation of growth hormone and the inhibition of luteinizing hormone observed after systemic administration of opiates ; 4) VIP inhibits SRIF release, by acting on VIP receptors present on MBH SRIF terminals ; the effect is consistent with the stimulation of GH reported after in vivo administration of the peptide.

In order to investigate mechanisms underlying the ovarian steroid action on hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, LHRH and a higher immunoreactive molecular form (MW 1,800 daltons) of the decapeptide were immunoassayed with antibodies of different specificities in hypothalamic subcellular fractions, after molecular sieve filtration on Biogel P4 columns equilibrated with 0.2 N acetic acid containing 0.02% sodium azide. The study was performed in ovariectomized (OVX), ovariectomized estradiol-implanted (OVX + E2) or OVX + E2 progesterone one-treated rats (OVX + E2 + P). The animals were killed before or during the circadian luteinizing hormone (LH) surge. The amount of LHRH-like immunoreactivity recovered from the synaptosomal fraction was slightly increased in OVX + E2-implanted animals but very markedly augmented in OVX + E2 + P-treated rats. In contrast, the higher molecular form recovered from a high-speed supernatant was markedly decreased in OVX + E2 + P-treated rats when compared to the other groups. At the time of maximal LH release induced by E2 + P administration, hypothalamic LHRH was markedly depleted, whereas the larger molecular form was notably augmented. The data suggest that ovarian steroids not only influence release of hypothalamic LHRH but also the processing of LHRH precursor forms.

The phasic luteinizing hormone (LH) release observed in ovariectomized (OVX), estrogen-implanted rats was further amplified and advanced when progesterone (P) was given 4 h prior to the gonadotropin surge. In contrast, an inhibitory effect of P on the daily LH surge was observed when P was administered 16-36 h prior to LH peak. In order to determine whether this biphasic action of P is primarily exerted on the release of luteinizing hormone releasing hormone (LHRH), on the pituitary response to LHRH, or on both, mediobasal hypothalamic slices or pituitary fragments of adult OVX rats or of OVX rats pretreated with estrogen alone or in combination with P were tested in a perifusion system. Mediobasal hypothalamic slices were perifused in buffered (pH 7.2) oxygenated Locke's medium containing bacitracin (2 X 10(-5) M). In the absence of estrogen pretreatment, high (56 mM) concentrations of K+ were barely effective in releasing LHRH. Subcutaneous implantation of 17 beta-estradiol for 5 days markedly increased the amplitude of the LHRH secretory response to K+ depolarization. Additional administration of P (25 mg/rat s.c.) 4 h before sacrifice further amplified the K+-induced LHRH release. In contrast, the K+-evoked LHRH secretion was significantly inhibited when P was given 16 or 36 h before. Estradiol thus appears to facilitate the LHRH secretory response to depolarizing stimuli, whereas P either enhances or blocks the induced LHRH release depending upon its time of administration. At the pituitary level, the sensitivity of LHRH-induced LH release was also increased after estrogen pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental increase in serotonin (5-HT) induced shortly before the "critical period" of ovulation control blocks the release of LH. Administration of a synthesis inhibitor of the amine p-chlorophenylalanine, (pCP) is ineffective when given during the critical period. However, pCP given in the evening of dioestrus II (18 to 24 hours before the "critical period") blocks ovulation. That 5-HT is specifically involved in this delayed effect of the drug is shown by the ability of increasing doses of 5-hydroxytryptophane (5-HTP) to induce with pCP a graded restoration of LH release when given together. This result, obtained in immature rats, has been confirmed in cyclic ones. A positive, permissive effect of 5-HT containing neurons on neural processes leading to LH release and ovulation is thus postulated. This action affects early stages of the LH release regulating mechanisms, in contrast to the classically described inhibiting effect of the amine, which blocks the actual release of LH-RH at the median eminence level during the critical period.

We previously demonstrated a specific stimulatory action of estrogens on phosphatidylethanolamine methylation in rat pituitary membranes. To investigate the physiological relevancy of this effect, the activity of methylating enzyme(s) was evaluated during the rat estrous cycle, a period in which both endogenous ovarian steroid levels and the sensitivity of pituitary membrane receptors fluctuate. Anterior pituitary membranes (P2) were prepared from adult female rats at different stages of the estrous cycle and assayed for phospholipid methylation in the presence of S-adenosyl-[methyl-3H]methionine as a donor of 3H-methyl groups. Methylated phospholipids were separated by TLC. Formation of phosphatidyl-mono- and dimethylethanolamine and that of phosphatidylcholine increased significantly in the morning, reaching maximal values on the afternoon of proestrus; they decreased thereafter during estrus, metestrus, and diestrus. Plasma estradiol concentrations increased in late diestrus and then varied similarly with the fluctuations of phospholipid methyltransferase activity throughout the cycle. In parallel, plasma levels of LH and PRL were significantly elevated during the afternoon of proestrus, but remained low throughout the rest of the cycle. Under the same experimental conditions, phospholipid methylation in membranes prepared from mediobasal-hypothalamic structures was not affected. These data demonstrate that under physiological conditions the increased pituitary methyltransferase activity is associated with the progressive increment of plasma estradiol levels occurring shortly before proestrus and precedes the release of LH and PRL. Ovariectomy significantly decreased methyltransferase activity; however, 17 beta-estradiol treatment of ovariectomized rats for 5 days restored the enzyme activity, which was further augmented after progesterone administration. Attempting to investigate variations of pituitary methyltransferase activity in male rats, we demonstrated that the intact males showed weaker activity than that of females; orchidectomy diminished the phospholipid methylation, but adrenalectomy had no effect.

17 beta-Estradiol (E2) affects the sensitivity of pituitary cells to several neurohormones as LHRH, TRH, or dopamine, presumably by modulating receptor coupling mechanisms. We attempted to pinpoint the membrane processes underlying this modulation and studied the effect of E2 on pituitary membrane phospholipid methylation. Anterior pituitary membranes prepared from ovariectomized (ovx) or ovx plus E2-treated rats were assayed for phospholipid methylation. Methylated phospholipids were separated by TLC. Incorporation of [3H]methyl groups into phospholipids increased with membrane concentration and incubation time with S-adenosyl-L-methyl [3H]methionine; it was not Mg2+ dependent and was inhibited in a dose-dependent manner by S-adenosyl-L-homocysteine, methyltransferase inhibitor. pH was found to be critical. Formation of phosphatidyl-monoethanolamine, phosphatidyl-dimethylethanolamine, and phosphatidylcholine was markedly stimulated by treatment with E2. The effect increased progressively when animals were killed 15 h to 5 days after E2 implantation. The response involved a shift in the maximum velocity (Vmax) although there was no change in the available substrate for the methylating enzyme. This change in Vmax probably reflects changes in the amount of the methylating enzyme itself. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2 was ineffective, pointing to a stereospecific interaction. After differential centrifugation of pituitary membranes, the highest specific methyltransferase activity was found in light mitochondrial (L) and microsomal (P) fractions and the lowest in nuclei (N) and the heavy mitochondrial (M) fractions. After sucrose density gradient centrifugation, methylated phospholipids were preferentially recovered from fractions corresponding to the endoplasmic reticulum and/or secretory granules. E2 treatment for 5 days did not modify the subcellular distribution of methyltransferase activity but stimulated it in all fractions; in contrast, it did not modify the activity of the other enzymes measured as fraction markers. Under the same experimental conditions, phospholipid methylation in membranes prepared from cortex, and anterior and mediobasal hypothalamic structures was not affected by the steroid, with the exception of a slight increment of [3H]methyl incorporation into mediobasal hypothalamic membrane phospholipids after 5 days of E2 treatment. These results indicate that E2-induced changes in pituitary responsiveness might be concomitant with selective effects of the steroid on specific membrane enzymatic activities involved in coupling mechanisms.

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 degrees C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 X 10(-5) M) was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 microM) or the ionophore A 23187 (10(-5) M), markedly stimulated the release of both neuropeptides. Trifluoperazine, a calmodulin inhibitor, decreased the K+-evoked LHRH and SRIF release in a dose-dependent manner; it was also effective in inhibiting the veratridine-induced neuropeptides release. Phenytoin, a calmodulin-dependent kinase inhibitor, also decreased in a dose-dependent manner the K+-induced LHRH and SRIF release; the basal release of both neuropeptides remained unaffected by either treatment. The ionophore-stimulated release of both neuropeptides was significantly inhibited as well. These data demonstrate that a Ca++-calmodulin kinase system may be involved in the mechanism of depolarization-induced LHRH and SRIF release from hypothalamic nerve terminals.