Chemogenetic activation of PVN CRH neurons disrupts the estrous cycle and LH dynamics in female mice

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Introduction: The impact of stress on reproductive function is significant. Hypothalamic paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) plays a major role in regulating the stress response. Understanding how the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis interact is crucial for comprehending how stress can lead to reproductive dysfunction. However, whether stress influences reproductive function via modulating PVN CRH or HPA sequelae is not fully elucidated. Methods: In this study, we investigated the impact of chemogenetic activation of PVN CRH neurons on reproductive function. We chronically and selectively stimulated PVN CRH neurons in female CRH-Cre mice using excitatory designer receptor exclusively activated by designer drugs (DREADDs) viral constructs, which were bilaterally injected into the PVN. The agonist compound-21 (C21) was delivered through the drinking water. We determined the effects of DREADDs activation of PVN CRH neurons on the estrous cycles, LH pulse frequency in diestrus and metestrus and LH surge in proestrus mice. The effect of long-term C21 administration on basal corticosterone secretion and the response to acute restraint stress during metestrus was also examined. Additionally, computer simulations of a mathematical model were used to determine the effects of DREADDs activation of PVN CRH neurons, simulating chronic stress, on the physiological parameters examined experimentally. Results: As a result, and consistent with our mathematical model predictions, the length of the estrous cycle was extended, with an increase in the time spent in estrus and metestrus, and a decrease in proestrus and diestrus. Additionally, the frequency of LH pulses during metestrus was decreased, but unaffected during diestrus. The occurrence of the preovulatory LH surge during proestrus was disrupted. The basal level of corticosterone during metestrus was not affected, but the response to acute restraint stress was diminished after long-term C21 application. Discussion: These data suggest that PVN CRH neurons play a functional role in disrupting ovarian cyclicity and the preovulatory LH surge, and that the activity of the GnRH pulse generator remains relatively robust during diestrus but not during metestrus under chronic stress exposure in accordance with our mathematical model predictions.Biotechnology and Biological Sciences Research Council (BBSRC)Engineering and Physical Sciences Research Council (EPSRC)Medical Research Council (MRC

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

Incentivizing the Use of Quantified Self Devices: The Cases of Digital Occupational Health Programs and Data-Driven Health Insurance Plans

Initially designed for a use in private settings, smartwatches, activity trackers and other quantified self devices are receiving a growing attention from the organizational environment. Firms and health insurance companies, in particular, are developing digital occupational health programs and data-driven health insurance plans centered around these systems, in the hope of exploiting their potential to improve individual health management, but also to gather large quantities of data. As individual participation in such organizational programs is voluntary, organizations often rely on motivational incentives to prompt engagement. Yet, little is known about the mechanisms employed in organizational settings to incentivize the use of quantified self devices. We therefore seek, in this exploratory paper, to offer a first structured overview of this topic and identify the main motivational incentives in two emblematical cases: digital occupational health programs and data-driven health insurance plans. By doing so, we aim to specify the nature of this new dynamic around the use of quantified self devices and define some of the key lines for further investigation

Inhaled corticosteroids and long-acting beta-agonists in adult asthma: a winning combination in all?

In the recent years, considerable insight has been gained in to the optimal management of adult asthma. Most adult patients with asthma have mild intermittent and persistent disease, and it is acknowledged that many patients do not reach full control of all symptoms and signs of asthma. Those with mild persistent asthma are usually not well controlled without inhaled corticosteroids (ICS). Studies have provided firm evidence that these patients can be well controlled when receiving ICS, especially when disease is of recent onset. This treatment should be given on a daily basis at a low dose and when providing a good response should be maintained to prevent severe exacerbations and disease deterioration. Intermittent ICS treatment at the time of an exacerbation has also been suggested as a strategy for mild persistent asthma, but it is less effective than low-dose regular treatment for most outcomes. Adding a long-acting beta-agonist (LABA) to ICS appears to be unnecessary in most of these patients for optimising control of their asthma. Patients with moderate persistent asthma can be regarded as those who are not ideally controlled on low-dose ICS alone. The combination of an ICS and LABA is preferred in these patients, irrespective of the brand of medicine, and this combination is better than doubling or even quadrupling the dose of ICS to achieve better asthma control and reduce exacerbation risks. An ICS/LABA combination in a single inhaler represents a safe, effective and convenient treatment option for the management of patients with asthma unstable on inhaled steroids alone. Ideally, once asthma is under full control, the dose of inhaled steroids should be reduced, which is possible in many patients. The duration of treatment before initiating this dose reduction has, however, not been fully established. One of the combinations available to treat asthma (budesonide and formoterol) has also been assessed as both maintenance and rescue therapy with a further reduction in the risk for a severe exacerbation. Clinical effectiveness in the real world now has to be established, since this approach likely improves compliance with regular maintenance therapy

Phase II study of second-line therapy with DTIC, BCNU, cisplatin and tamoxifen (Dartmouth regimen) chemotherapy in patients with malignant melanoma previously treated with dacarbazine

This study assessed response rates to combination dacarbazine (DTIC), BCNU (carmustine), cisplatin and tamoxifen (DBPT) chemotherapy in patients with progressive metastatic melanoma previously treated with DTIC, as an evaluation of DBPT as a second-line regimen, and as an indirect comparison of DBPT with DTIC. Thirty-five consecutive patients received DBPT. The patients were divided into two groups. Group 1 comprised 17 patients with progressive disease (PD) on DTIC + tamoxifen therapy who were switched directly to DBPT. Group 2 comprised 18 patients not immediately switched to DBPT and included patients who had either a partial response (PR; one patient) or developed stable disease (SD; four patients) with DTIC, or received adjuvant DTIC (nine patients). All except four patients had received tamoxifen at the time of initial DTIC treatment. Median times since stopping DTIC were 22 days (range 20–41) and 285 days (range 50–1240) in Groups 1 and 2 respectively. In Group 1, one patient developed SD for 5 months and the remainder had PD. In Group 2, there were two PRs, four patients with SD (4, 5, 6, and 6 months), and 11 with PD. These results indicate that the DBPT regimen is not of value in melanoma primarily refractory to DTIC. There were responses in patients not directly switched from DTIC to DBPT, suggesting combination therapy may be of value in a small subgroup of melanoma patients. © 2000 Cancer Research Campaig

KRAS-mutation incidence and prognostic value are metastatic site-specific in lung adenocarcinoma: poor prognosis in patients with KRAS-mutation and bone metastasis

Current guidelines lack comprehensive information on the metastatic site-specific role of KRAS mutation in lung adenocarcinoma (LADC). We investigated the effect of KRAS mutation on overall survival (OS) in this setting. In our retrospective study, 500 consecutive Caucasian metastatic LADC patients with known KRAS mutational status were analyzed after excluding 32 patients with EGFR mutations. KRAS mutation incidence was 28.6%. The most frequent metastatic sites were lung (45.6%), bone (26.2%), adrenal gland (17.4%), brain (16.8%), pleura (15.6%) and liver (11%). Patients with intrapulmonary metastasis had significantly increased KRAS mutation frequency compared to those with extrapulmonary metastases (35% vs 26.5%, p=0.0125). In contrast, pleural dissemination and liver involvement were associated with significantly decreased KRAS mutation incidence (vs all other metastatic sites; 17% (p<0.001) and 16% (p=0.02) vs 33%, respectively). Strikingly, we found a significant prognostic effect of KRAS status only in the bone metastatic subcohort (KRAS-wild-type vs KRAS-mutant; median OS 9.7v 3.7 months; HR, 0.49; 95% CI, 0.31 to 0.79; p =0.003). Our study suggests that KRAS mutation frequency in LADC patients shows a metastatic site dependent variation and, moreover, that the presence of KRAS mutation is associated with significantly worse outcome in bone metastatic cases.(VLID)469049

Stereodivergent Synthesis of Enantioenriched 4-Hydroxy-2- cyclopentenones

Protected 4-hydroxycyclopentenones (4-HCPs) constitute an important class of intermediates in chemical synthesis. A route to this class of compound has been developed. Key steps include Noyori reduction (which establishes the stereochemistry of the product), ring-closing metathesis, and simple functional group conversions to provide a set of substituted 4-HCPs in either enantiomeric form

Striatal interneurons in dissociated cell culture

In addition to the well-characterized direct and indirect projection neurons there are four major interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. It might be assumed that dissociated cell cultures of striatum (typically from embryonic day E18.5 in rat and E14.5 for mouse) contain each of these neuronal types. However, in dissociated rat striatal (caudate/putamen, CPu) cultures arguably the most important interneuron, the giant aspiny cholinergic neuron, is not present. When dissociated striatal neurons from E14.5 Sprague–Dawley rats were mixed with those from E18.5 rats, combined cultures from these two gestational periods yielded surviving cholinergic interneurons and representative populations of the other interneuron types at 5 weeks in vitro. Neurons from E12.5 CD-1 mice were combined with CPu neurons from E14.5 mice and the characteristics of striatal interneurons after 5 weeks in vitro were determined. All four major classes of interneurons were identified in these cultures as well as rare tyrosine hydroxylase positive interneurons. However, E14.5 mouse CPu cultures contained relatively few cholinergic interneurons rather than the nearly total absence seen in the rat. A later dissection day (E16.5) was required to obtain mouse CPu cultures totally lacking the cholinergic interneuron. We show that these cultures generated from two gestational age cells have much more nearly normal proportions of interneurons than the more common organotypic cultures of striatum. Interneurons are generated from both ages of embryos except for the cholinergic interneurons that originate from the medial ganglionic eminence of younger embryos. Study of these cultures should more accurately reflect neuronal processing as it occurs in the striatum in vivo. Furthermore, these results reveal a procedure for parallel culture of striatum and cholinergic depleted striatum that can be used to examine the function of the cholinergic interneuron in striatal networks

Phase II study of the oxygen saturation curve left shifting agent BW12C in combination with the hypoxia activated drug mitomycin C in advanced colorectal cancer

BW12C (5-[2-formyl-3-hydroxypenoxyl] pentanoic acid) stabilizes oxyhaemoglobin, causing a reversible left-shift of the oxygen saturation curve (OSC) and tissue hypoxia. The activity of mitomycin C (MMC) is enhanced by hypoxia. In this phase II study, 17 patients with metastatic colorectal cancer resistant to 5-fluorouracil (5-FU) received BW12C and MMC. BW12C was given as a bolus loading dose of 45 mg kg−1over 1 h, followed by a maintenance infusion of 4 mg kg−1h−1for 5 h. MMC 6 mg m−2was administered over 15 min immediately after the BW12C bolus. The 15 evaluable patients had progressive disease after a median of 2 (range 1–4) cycles of chemotherapy. Haemoglobin electrophoresis 3 and 5 h after the BW12C bolus dose showed a fast moving band consistent with the BW12C-oxyhaemoglobin complex, accounting for approximately 50% of total haemoglobin. The predominant toxicities – nausea/vomiting and vein pain – were mild and did not exceed CTC grade 2. Liver31P magnetic resonance spectroscopy of patients with hepatic metastases showed no changes consistent with tissue hypoxia. The principle of combining a hypoxically activated drug with an agent that increases tissue hypoxia is clinically feasible, producing an effect equivalent to reducing tumour oxygen delivery by at least 50%. However, BW12C in combination with MMC for 5-FU-resistant colorectal cancer is not an effective regimen. This could be related to drug resistance rather than a failure to enhance cytotoxicity. © 2000 Cancer Research Campaig