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Kv3.1 channels are voltage-gated potassium channels that enable fast repolarization of the neuronal action potential, and are essential for the high-frequency, high-fidelity firing of neurons in the auditory brainstem and midbrain. Altered activity of these neurons has been implicated in the generation of tinnitus induced by noise exposure. Furthermore, loss of Kv3 channel function has been observed shortly after noise exposure, which may contribute to the maladaptive plasticity leading to the emergence of tinnitus. AUT00063 is a novel, selective modulator of Kv3.1 channels that has been investigated in a number of animal models of noise-induced hearing loss and tinnitus. In Long-Evans rats exposed to a unilateral 116 dB, 16 kHz octave-band noise for one hour, approximately half of the animals demonstrated deficits in auditory gap processing, consistent with the presence of tinnitus. Acute treatment with AUT00063 abolished evidence of tinnitus, without affecting the behavior of control animals or noise-exposed animals without tinnitus. In CBA/Ca mice exposed to 8-16 kHz noise at 105 dB SPL for two hours under anaesthesia, there was a significant increase in spontaneous activity of neurons in the inferior colliculus (IC) when recorded 4 weeks later. AUT00063 decreased IC excitability in noise-exposed animals, returning spontaneous rates to levels similar to those observed in control animals; no such effect was observed following injections of vehicle. These data support the evaluation of AUT00063 for the treatment of people with subjective, noise-related tinnitus. Funding and disclosure: These studies were financially supported by Autifony Therapeutics Limited. CHL, NP and GSA are full time employees of Autifony Therapeutics Limited or Autifony SRL. [ABSTRACT FROM AUTHOR]

Animal research facilities are noisy environments. The high air change rates required in animal housing spaces tend to create higher noise levels from the heating and cooling systems. Housing rooms are typically constructed of hard wall material that is easily cleaned but simultaneously highly reverberant, meaning that the sound cannot be controlled/attenuated so the sounds that are generated bounce around the room uncontrolled. (Soft, sound-absorbing surfaces generally cannot be used in animal facilities because they collect microbes; various wall surface features and sound control panel options are available, although rarely used.) In addition, many of our husbandry tasks such as cage changing, animal health checks, cleaning, and transporting animals produce high levels of noise. Finally, much of the equipment we have increasingly employed in animal housing spaces, such as ventilated caging motors, biosafety, or procedure cabinets, can generate high levels of background noise, including ultrasound. These and many additional factors conspire to create an acoustic environment that is neither naturalistic nor conducive to a stress-free environment. The acoustic variability both within and between institutions can serve as an enormous confounder for research studies and a threat to our ability to reproduce studies over time and between research laboratories. By gaining a better appreciation for the acoustic variables, paired with transparency in reporting the levels, we might be able to gain a better understanding of their impacts and thereby gain some level of control over such acoustic variables in the animal housing space. The result of this could improve both animal welfare and study reproducibility, helping to address our 3Rs goals of Replacement, Reduction, and Refinement in the animal biomedical research enterprise.

To investigate the symptoms of SARS-CoV-2 infection, their dynamics and their discriminatory power for the disease using longitudinally, prospectively collected information reported at the time of their occurrence. We have analysed data from a large phase 3 clinical UK COVID-19 vaccine trial. The alpha variant was the predominant strain. Participants were assessed for SARS-CoV-2 infection via nasal/throat PCR at recruitment, vaccination appointments, and when symptomatic. Statistical techniques were implemented to infer estimates representative of the UK population, accounting for multiple symptomatic episodes associated with one individual. An optimal diagnostic model for SARS-CoV-2 infection was derived. The 4-month prevalence of SARS-CoV-2 was 2.1%; increasing to 19.4% (16.0%-22.7%) in participants reporting loss of appetite and 31.9% (27.1%-36.8%) in those with anosmia/ageusia. The model identified anosmia and/or ageusia, fever, congestion, and cough to be significantly associated with SARS-CoV-2 infection. Symptoms' dynamics were vastly different in the two groups; after a slow start peaking later and lasting longer in PCR+ participants, whilst exhibiting a consistent decline in PCR- participants, with, on average, fewer than 3 days of symptoms reported. Anosmia/ageusia peaked late in confirmed SARS-CoV-2 infection (day 12), indicating a low discrimination power for early disease diagnosis.

Background: The recombinant protein-based vaccine, NVX-CoV2373, demonstrated 89.7% efficacy against coronavirus disease 2019 (COVID-19) in a phase 3, randomized, observer-blinded, placebo-controlled trial in the United Kingdom. The protocol was amended to include a blinded crossover. Data to the end of the placebo-controlled phase are reported., Methods: Adults aged 18-84 years received 2 doses of NVX-CoV2373 or placebo (1:1) and were monitored for virologically confirmed mild, moderate, or severe COVID-19 (onset from 7 days after second vaccination). Participants who developed immunoglobulin G (IgG) against nucleocapsid protein but did not show symptomatic COVID-19 were considered asymptomatic. Secondary outcomes included anti-spike (S) IgG responses, wild-type virus neutralization, and T-cell responses., Results: Of 15 185 participants, 13 989 remained in the per-protocol efficacy population (6989 NVX-CoV2373, 7000 placebo). At a maximum of 7.5 months (median, 4.5) postvaccination, there were 24 cases of COVID-19 among NVX-CoV2373 recipients and 134 cases among placebo recipients, a vaccine efficacy of 82.7% (95% confidence interval [CI], 73.3%-88.8%). Vaccine efficacy was 100% (95% CI, 17.9%-100.0%) against severe disease and 76.3% (95% CI, 57.4%-86.8%) against asymptomatic disease. High anti-S and neutralization responses to vaccination were evident, together with S-protein-specific induction of interferon-γ secretion in peripheral blood T cells. Incidence of serious adverse events and adverse events of special interest were similar between groups., Conclusions: A 2-dose regimen of NVX-CoV2373 conferred a high level of ongoing protection against asymptomatic, symptomatic, and severe COVID-19 through >6 months postvaccination. A gradual decrease of protection suggests that a booster may be indicated., Clinical Trials Registration: EudraCT, 2020-004123-16., Competing Interests: Potential conflicts of interest. K. A., I. C., L. F., G. G., I. Mc., E. J. R., A. R., K. S., and S. T. are employees of Novavax Inc and receive a salary for their work. K. S. reports stock received as part of employment compensation from Novavax. A. R. reports stock or stock options from Novavax. E. J. R. and I. Mc. report Novavax stock. K. A. reports vested and unvested Novavax stock/restricted stock units. I. C. reports Novavax stock and stock options and salary and bonus as an employee of Novavax. L. F. reports consulting fees as a prior full-time employee, now contractor to, Novavax reimbursed hourly for work performed on this study and in analyses and drafting this report, and shares and stock options from Novavax. G. G. reports stock-related compensations from Novavax. S. T. reports royalties or licenses, salary and stock, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events, and a leadership or fiduciary role in other board, society, committee, or advocacy group, paid or unpaid, as an employee of Novavax. Guy's and St Thomas' National Health Service (NHS) Foundation Trust (with which A. L. G. is affiliated) received funding from Novavax for this trial. Oxford University has entered into a partnership with AstraZeneca for further development of ChAdOx1 nCoV-19. A. L. G. is named as an inventor on a patent covering use of a particular promoter construct that is often used in vectored vaccines and is incorporated in the ChAdOx1 nCoV-19 vaccine, and may benefit from royalty income paid to the University of Oxford from sales of this vaccine by AstraZeneca and its sublicensees under the university's revenue sharing policy. M. B. reports a research grant to institution from Novavax related to this manuscript, advisory/speaker fees or grants to the institution from GSK, ViiV, Gilead, Janssen, Moderna, Pfizer, Valneva, MSD, Roche, Cipla, and Mylan and support for attending the online World AIDS conference from ViiV. D. R. C. reports a research grant to institution from Gilead Sciences, unpaid participation as an Independent Data Monitoring Board (IDMB) member for the FLARE Trial, and unpaid role as a British HIV Association trustee member. J. G. reports a research contract with institution from Novavax. C. A. C. reports a research grant to institution from Novavax related to this manuscript, and a research grant to institution from Moderna. P. T. H. reports a research grant to institution from Novavax related to this manuscript, research grants to institution from Pfizer, AstraZeneca, Moderna, Valneva, and Janssen, and payment to institution for educational events from Novavax. P. A. K. reports a research grant to institution from Novavax related to this manuscript, grants or contracts unrelated to this work from Vifor, Astellas, Evotec, Pharmacosmos, and Unicyte; consulting fees from AstraZeneca, Vifor, Unicyte, and UCB; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Vifor, AstraZeneca, Pfizer, Pharmacosmos, Napp, and Bayer; and support for attending meetings and/or travel from Pharmacosmos and Vifor. J. P. reports a research grant to institution from Novavax related to this manuscript, and being co-applicant for a Haywood Foundation grant and for a National Institute for Health and Care Research (NIHR)/Clinical Research Network (CRN) COVID Innovation and Insight grant. P. A. S. reports a research grant to institution from Novavax related to this manuscript, payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Bayer and AstraZeneca; support for attending meetings and/or travel from Bayer; and participation on a DSMB or advisory board for Bayer. E. C. T. reports a research grant to institution from Novavax related to this manuscript, research grants to institution from Valneva, COV-BOOST, Medical Research Council (MRC), Wellcome, and Public Health Scotland; payment to author for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Wellcome Connecting Science—Sanger Institute; support for attending meetings and/or travel, paid to author, from Wellcome Connecting Science—Sanger Institute, University of Oxford, University of Cambridge, and University of Manchester; and unpaid leadership or fiduciary roles with the Scottish Committee for Pandemic Preparedness, UK Health Security Agency (HSA) technical groups, and the Scottish Genomics Oversight Group. J. T. reports Quin Technologies consulting fees; participation as chair of the Data Monitoring and Ethics Committee (DMEC) for the NIFTY Trial, an NIHR-funded trial; and a role as clinical advisor to Parathyroid UK, a patient support charity. S. I., C. J., H. N., D. B., A. Hi., R. S., I. M., A. M., R. C., D. N. B., D. S., E. P. G., J. B., R. L. S., A. He., C. M., C. P., and F. B. report a research grant to institution from Novavax related to this manuscript. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objectives: Individuals with chronic hypoparathyroidism may experience suboptimal medical care with high frequency of unplanned hospitalisation and iatrogenic harm. In 2015 the European Society for Endocrinology published consensus guidelines on the management of chronic hypoparathyroidism. We set out to audit compliance with these guidelines., Methods: Using these recommendations as audit standards we worked with the Society for Endocrinology and Parathyroid UK to conduct a national audit of management of chronic hypoparathyroidism in the United Kingdom. Endocrine leads in 117 endocrine departments were invited to participate in the survey by completing a data collection tool on up to 5 sequential cases of chronic hypoparathyroidism seen in their outpatient clinics in the preceding 12 months. Data were collected on 4 treatment standards and 9 monitoring standards. Data on hospitalisations and Quality of Life monitoring were also collected., Results: Responses were received from 22 departments giving a response rate of 19%, concerning 80 individual cases. The mean age of subjects was 48.4 years. The main findings were that the commonest cause of hypoparathyroidism was post surgical (66.3%). Treatments taken by the group included activated vitamin D analogues (96.3%), oral calcium salts (66.3%), vitamin D supplements (17.5%), thiazide diuretics (5%) and rhPTH 1-34 (1.3%). Compliance with the audit standards varied between 98.8% and 60% for the treatment standards and between 91.3% and 20% for the monitoring standards. Some of the areas of weakness revealed include low rates of 24 h urinary calcium excretion monitoring, serum magnesium monitoring and low rates of renal imaging where indicated. In addition and importantly, 16.3% of subjects had experienced at least one hospital admission in the preceding 12 months., Conclusion: We conclude that further improvements in the UK national standard of management of chronic hypoparathyroidism should be made and that this will benefit both quality of life, morbidity and potentially mortality in this group of patients., (© 2022 The Authors. Clinical Endocrinology published by John Wiley & Sons Ltd.)

Background: Early clinical data from studies of the NVX-CoV2373 vaccine (Novavax), a recombinant nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that contains the full-length spike glycoprotein of the prototype strain plus Matrix-M adjuvant, showed that the vaccine was safe and associated with a robust immune response in healthy adult participants. Additional data were needed regarding the efficacy, immunogenicity, and safety of this vaccine in a larger population., Methods: In this phase 3, randomized, observer-blinded, placebo-controlled trial conducted at 33 sites in the United Kingdom, we assigned adults between the ages of 18 and 84 years in a 1:1 ratio to receive two intramuscular 5-μg doses of NVX-CoV2373 or placebo administered 21 days apart. The primary efficacy end point was virologically confirmed mild, moderate, or severe SARS-CoV-2 infection with an onset at least 7 days after the second injection in participants who were serologically negative at baseline., Results: A total of 15,187 participants underwent randomization, and 14,039 were included in the per-protocol efficacy population. Of the participants, 27.9% were 65 years of age or older, and 44.6% had coexisting illnesses. Infections were reported in 10 participants in the vaccine group and in 96 in the placebo group, with a symptom onset of at least 7 days after the second injection, for a vaccine efficacy of 89.7% (95% confidence interval [CI], 80.2 to 94.6). No hospitalizations or deaths were reported among the 10 cases in the vaccine group. Five cases of severe infection were reported, all of which were in the placebo group. A post hoc analysis showed an efficacy of 86.3% (95% CI, 71.3 to 93.5) against the B.1.1.7 (or alpha) variant and 96.4% (95% CI, 73.8 to 99.5) against non-B.1.1.7 variants. Reactogenicity was generally mild and transient. The incidence of serious adverse events was low and similar in the two groups., Conclusions: A two-dose regimen of the NVX-CoV2373 vaccine administered to adult participants conferred 89.7% protection against SARS-CoV-2 infection and showed high efficacy against the B.1.1.7 variant. (Funded by Novavax; EudraCT number, 2020-004123-16.)., (Copyright © 2021 Massachusetts Medical Society.)

Background: The purpose of this trial was to test if the Norfolk Diabetes Prevention Study (NDPS) lifestyle intervention, recently shown to reduce the incidence of type 2 diabetes in high-risk groups, also improved glycaemic control in people with newly diagnosed screen-detected type 2 diabetes., Methods: We screened 12,778 participants at high risk of type 2 diabetes using a fasting plasma glucose and glycosylated haemoglobin (HbA1c). People with screen-detected type 2 diabetes were randomised in a parallel, three-arm, controlled trial with up to 46 months of follow-up, with a control arm (CON), a group-based lifestyle intervention of 6 core and up to 15 maintenance sessions (INT), or the same intervention with additional support from volunteers with type 2 diabetes trained to co-deliver the lifestyle intervention (INT-DPM). The pre-specified primary end point was mean HbA1c compared between groups at 12 months., Results: We randomised 432 participants (CON 149; INT 142; INT-DPM 141) with a mean (SD) age of 63.5 (10.0) years, body mass index (BMI) of 32.4 (6.4) kg/m 2 , and HbA1c of 52.5 (10.2) mmol/mol. The primary outcome of mean HbA1c at 12 months (CON 48.5 (9.1) mmol/mol, INT 46.5 (8.1) mmol/mol, and INT-DPM 45.6 (6.0) mmol/mol) was significantly lower in the INT-DPM arm compared to CON (adjusted difference -2.57 mmol/mol; 95% CI -4.5, -0.6; p = 0.007) but not significantly different between the INT-DPM and INT arms (-0.55 mmol/mol; 95% CI -2.46, 1.35; p = 0.57), or INT vs CON arms (-2.14 mmol/mol; 95% CI -4.33, 0.05; p = 0.07). Subgroup analyses showed the intervention had greater effect in participants < 65 years old (difference in mean HbA1c compared to CON -4.76 mmol/mol; 95% CI -7.75, -1.78 mmol/mol) than in older participants (-0.46 mmol/mol; 95% CI -2.67, 1.75; interaction p = 0.02). This effect was most significant in the INT-DPM arm (-6.01 mmol/mol; 95% CI -9.56, -2.46 age < 65 years old and -0.22 mmol/mol; 95% CI -2.7, 2.25; aged > 65 years old; p = 0.007). The use of oral hypoglycaemic medication was associated with a significantly lower mean HbA1c but only within the INT-DPM arm compared to CON (-7.0 mmol/mol; 95% CI -11.5, -2.5; p = 0.003)., Conclusion: The NDPS lifestyle intervention significantly improved glycaemic control after 12 months in people with screen-detected type 2 diabetes when supported by trained peer mentors with type 2 diabetes, particularly those receiving oral hypoglycaemics and those under 65 years old. The effect size was modest, however, and not sustained at 24 months., Trial Registration: ISRCTN34805606 . Retrospectively registered 14.4.16., (© 2021. The Author(s).)

Hypocalcaemia is a frequent, and potentially dangerous complication of total thyroidectomy occurring secondary to devascularisation of the parathyroid glands. This quality improvement (QI) project was undertaken in a large Ear, Nose and Throat (ENT) department in the East of England over a one year period. The project aimed to improve postoperative guideline compliance by optimising the recognition and management of patients at risk of hypocalcaemia. This process focussed on improving parathyroid hormone (PTH) and calcium blood testing, prophylactic calcium prescribing and the subsequent monitoring and management of hypocalcaemia. A baseline audit was conducted to determine the initial guideline compliance. The QI process subsequently involved the introduction of a new intraoperative PTH pathway and the amendment of trust guidelines. In addition, there was a focus on improving clinician awareness of guidelines, junior doctor education, communication between operating surgeons and junior doctors and the optimisation of patient handover. The project saw a significant improvement in the monitoring of hypocalcaemia (from 22.2% to 83.3% for patients with an intermediate risk of hypocalcaemia) and in the prescribing of prophylactic calcium supplements from 7.5% to 43.5%. The measurement of PTH at four hours improved from 42.5% to 52.2%. By optimising postoperative care, this QI project improved patient safety as well as impacting on the duration, and overall cost, of inpatient stay., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2021, Collins et al.)

Noise and vibration are present in every room of laboratory animal vivaria, with great variability from room-to-room and facility-to-facility. Such stimuli are rarely measured. As a result, the many stakeholders involved in biomedical research, (for example, funding agencies, construction personnel, equipment manufacturers, animal facility administrators, veterinarians, technicians, and scientists) have little awareness of the effects such stimuli may have on their research animals. Noise and vibration present a potential source of unrecognized animal distress, and a significant, uncontrolled and confounding variable in scientific studies. Unmeasured and unrecognized noise and vibration can therefore undermine the fundamental goals of the 3R's to refine animal models and reduce the number of animals used in biomedical and behavioral research. This overview serves to highlight the scope of this problem and proposes a series of recommended practices to limit its negative effects on research animals and the scientific data derived from them. These practices consist of developing a written plan for managing noise and vibration concerns, assessment of noise and vibration both annually and whenever unexpected changes in the facility or animals are observed, and for maintaining levels of chronic noise below thresholds that might cause animal welfare concerns or disruptions in ongoing studies.

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y 12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7-9 donors), with a potency ranking ADP (EC 50 1.3 ± 1.0 μM) > ATP (EC 50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y 2 receptor antagonism (AR-C118925XX; IC 50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y 6 receptor antagonist, MRS2587 (IC 50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y 2 and P2Y 6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.

White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic signalling and constitutive P2Y 2 receptor activation suppresses basal lipolysis in primary human in vitro -differentiated adipocytes. We found that human adipocytes possess cytoplasmic Ca 2+ tone due to ATP secretion and constitutive P2Y 2 receptor activation. Pharmacological antagonism or knockdown of P2Y 2 receptors increases intracellular cAMP levels and enhances basal lipolysis. P2Y 2 receptor antagonism works synergistically with phosphodiesterase inhibitors in elevating basal lipolysis, but is dependent upon adenylate cyclase activity. Mechanistically, we suggest that the increased Ca 2+ tone exerts an anti-lipolytic effect by suppression of Ca 2+ -sensitive adenylate cyclase isoforms. We also observed that acute enhancement of basal lipolysis following P2Y 2 receptor antagonism alters the profile of secreted adipokines leading to longer-term adaptive decreases in basal lipolysis. Our findings demonstrate that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling in human adipocytes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Leukocytes sense extracellular ATP, a danger-associated molecular pattern, released during cellular stress and death, via activation of cell surface P2X and P2Y receptors. Here, we investigate P2 receptor expression in primary human monocyte-derived macrophages and receptors that mediate ATP-evoked intracellular [Ca 2+ ] i signals and cytokine production in response to ATP concentrations that exclude P2X 7 receptor activation. Expression of P2X 1 , P2X 4 , P2X 5 , P2X 7 , P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , and P2Y 13 was confirmed by quantitative RT-PCR and immunocytochemistry. ATP elicited intracellular Ca 2+ responses in a concentration-dependent fashion (EC 50 = 11.4 ± 2.9 μM, n = 3). P2Y 11 and P2Y 13 activations mediated the amplitude of [Ca 2+ ] i response, whereas P2X 4 activation, but not P2X 1 or P2X 7 , determined the duration of Ca 2+ response during a sustained phase. ATP mediated gene induction of CXCL5, a proinflammatory chemokine. P2X 4 antagonism (PSB-12062 or BX430) inhibited ATP-mediated induction of CXCL5 gene expression and secretion of CXCL5 by primary macrophage. Inhibition of CXCL5 secretion by P2X 4 antagonists was lost in the absence of extracellular Ca 2+ Reciprocally, positive allosteric modulation of P2X 4 (ivermectin) augmented ATP-mediated CXCL5 secretion. P2X 7 , P2Y 11 , or P2Y 13 receptor did not contribute to CXCL5 secretion. Together, the data reveals a role for P2X 4 in determining the duration of ATP-evoked Ca 2+ responses and CXCL5 secretion in human primary macrophage., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Despite currently available therapies, most patients diagnosed with acute myeloid leukemia (AML) die of their disease. Tumor-host interactions are critical for the survival and proliferation of cancer cells; accordingly, we hypothesize that specific targeting of the tumor microenvironment may constitute an alternative or additional strategy to conventional tumor-directed chemotherapy. Because adipocytes have been shown to promote breast and prostate cancer proliferation, and because the bone marrow adipose tissue accounts for up to 70% of bone marrow volume in adult humans, we examined the adipocyte-leukemia cell interactions to determine if they are essential for the growth and survival of AML. Using in vivo and in vitro models of AML, we show that bone marrow adipocytes from the tumor microenvironment support the survival and proliferation of malignant cells from patients with AML. We show that AML blasts alter metabolic processes in adipocytes to induce phosphorylation of hormone-sensitive lipase and consequently activate lipolysis, which then enables the transfer of fatty acids from adipocytes to AML blasts. In addition, we report that fatty acid binding protein-4 (FABP4) messenger RNA is upregulated in adipocytes and AML when in coculture. FABP4 inhibition using FABP4 short hairpin RNA knockdown or a small molecule inhibitor prevents AML proliferation on adipocytes. Moreover, knockdown of FABP4 increases survival in Hoxa9/Meis1-driven AML model. Finally, knockdown of carnitine palmitoyltransferase IA in an AML patient-derived xenograft model improves survival. Here, we report the first description of AML programming bone marrow adipocytes to generate a protumoral microenvironment., (© 2017 by The American Society of Hematology.)

Fischer Brown Norway (FBN) rats (n = 233) were unilaterally exposed to 12 different combinations of noise intensity, duration, and spectrum, while 46 rats served as sham-exposed controls. Rats were behaviorally tested for tinnitus and hyperacusis using gap-induced inhibition of the acoustic startle reflex (Gap) and prepulse inhibition (PPI) using 60-dB SPL before noise-exposure and at regular intervals for 12 mo. 12-mo after noise exposure the middle-aged rats were then tested again for tinnitus and hyperacusis before collecting Auditory Brainstem Response (ABR) thresholds. Collapsing across all noise exposure conditions a significant tinnitus-like deficit in responding to silent gaps was observed, with the most likely tinnitus pitch around 16 kHz. Rates of tinnitus 12-mo after noise exposure were greatest in groups receiving the four least intense noise doses (110-dB for 30, 60 and 120 min, and 116-dB for 30 min), while some of the greatest rates of hyperacusis occurred in groups receiving more intense or longer exposures. The results suggest that rates for developing tinnitus in animal models may not be easily predicted based upon noise exposure dose, but that low-to-moderate noise exposures may result in the greatest likelihood for producing tinnitus., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests a role for inhibitory neurotransmitter dysfunction in the pathology of tinnitus. Opposing hypotheses proposed either a pathologic decrease or increase of GABAergic inhibition in medial geniculate body (MGB). In thalamus, GABA mediates fast synaptic inhibition via synaptic GABAA receptors (GABAARs) and persistent tonic inhibition via high-affinity extrasynaptic GABAARs. Given that extrasynaptic GABAARs control the firing mode of thalamocortical neurons, we examined tonic GABAAR currents in MGB neurons in vitro, using the following three groups of adult rats: unexposed control (Ctrl); sound exposed with behavioral evidence of tinnitus (Tin); and sound exposed with no behavioral evidence of tinnitus (Non-T). Tonic GABAAR currents were evoked using the selective agonist gaboxadol. Months after a tinnitus-inducing sound exposure, gaboxadol-evoked tonic GABAAR currents showed significant tinnitus-related increases contralateral to the sound exposure. In situ hybridization studies found increased mRNA levels for GABAAR δ-subunits contralateral to the sound exposure. Tin rats showed significant increases in the number of spikes per burst evoked using suprathreshold-injected current steps. In summary, we found little evidence of tinnitus-related decreases in GABAergic neurotransmission. Tinnitus and chronic pain may reflect thalamocortical dysrhythmia, which results from abnormal theta-range resonant interactions between thalamus and cortex, due to neuronal hyperpolarization and the initiation of low-threshold calcium spike bursts (Walton and Llinás, 2010). In agreement with this hypothesis, we found tinnitus-related increases in tonic extrasynaptic GABAAR currents, in action potentials/evoked bursts, and in GABAAR δ-subunit gene expression. These tinnitus-related changes in GABAergic function may be markers for tinnitus pathology in the MGB., (Copyright © 2015 the authors 0270-6474/15/359369-12$15.00/0.)

In addition to their extended lifespans, slow-aging growth hormone receptor/binding protein gene-disrupted (knockout) (GHR-KO) mice are hypoinsulinemic and highly sensitive to the action of insulin. It has been proposed that this insulin sensitivity is important for their longevity and increased healthspan. We tested whether this insulin sensitivity of the GHR-KO mouse is necessary for its retarded aging by abrogating that sensitivity with a transgenic alteration that improves development and secretory function of pancreatic β-cells by expressing Igf-1 under the rat insulin promoter 1 (RIP::IGF-1). The RIP::IGF-1 transgene increased circulating insulin content in GHR-KO mice, and thusly fully normalized their insulin sensitivity, without affecting the proliferation of any non-β-cell cell types. Multiple (nonsurvivorship) longevity-associated physiological and endocrinological characteristics of these mice (namely beneficial blood glucose regulatory control, altered metabolism, and preservation of memory capabilities) were partially or completely normalized, thus supporting the causal role of insulin sensitivity for the decelerated senescence of GHR-KO mice. We conclude that a delayed onset and/or decreased pace of aging can be hormonally regulated., (© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

Tinnitus has been associated with increased spontaneous and evoked activity, increased neural synchrony, and reorganization of tonotopic maps of auditory nuclei. However, the neurotransmitter systems mediating these changes are poorly understood. Here, we developed an in vitro assay that allows us to evaluate the roles of excitation and inhibition in determining the neural correlates of tinnitus. To measure the magnitude and spatial spread of evoked circuit activity, we used flavoprotein autofluorescence (FA) imaging, a metabolic indicator of neuronal activity. We measured FA responses after electrical stimulation of glutamatergic axons in slices containing the dorsal cochlear nucleus, an auditory brainstem nucleus hypothesized to be crucial in the triggering and modulation of tinnitus. FA imaging in dorsal cochlear nucleus brain slices from mice with behavioral evidence of tinnitus (tinnitus mice) revealed enhanced evoked FA response at the site of stimulation and enhanced spatial propagation of FA response to surrounding sites. Blockers of GABAergic inhibition enhanced FA response to a greater extent in control mice than in tinnitus mice. Blockers of excitation decreased FA response to a similar extent in tinnitus and control mice. These findings indicate that auditory circuits in mice with behavioral evidence of tinnitus respond to stimuli in a more robust and spatially distributed manner because of a decrease in GABAergic inhibition.

The nucleotide substitution C797T in the Chrm2 gene causes substitution of leucine for proline at position 266 (P266L) of the CHRM2 protein. Because Chrm2 codes for the type 2 muscarinic receptor, this mutation could influence physiologic and behavioral phenotypes of mice. Chrm2 mRNA was not differentially expressed in 2 brain regions with high cholinergic innervation in a mouse strain that does (BALB/cByJ) or does not (C57BL/6J) have the mutation. In addition, strains of mice with and without the C797T point mutation in Chrm2 did not differ significantly in muscarinic binding properties. Variation across strains was detected in terms of acoustic startle, prepulse inhibition, and the physiologic effects of the muscarinic agonist oxotremorine. However, interstrain differences in these measures did not correlate with the presence of the mutation. Although we were unable to associate a measurable phenotype with the Chrm2 mutation, assessment of the mutation on other genetic backgrounds or in the context of other traits might reveal differential effects. Therefore, despite our negative findings, evaluation of characteristics that involve muscarinic function should be undertaken with caution when comparing mice with different alleles of the Chrm2 gene.

Presbycusis can be considered a slow age-related peripheral and central deterioration of auditory function which manifests itself as deficits in speech comprehension, especially in noisy environments. The present study examined neural correlates of a simple broadband noise stimulus in primary auditory cortex (A1) of young and aged Fischer-Brown Norway (FBN) rats. Age-related changes in unit responses to broadband noise bursts and spontaneous activity were simultaneously recorded across A1 layers using a single shank, 16-channel electrode. Noise bursts were presented contralateral to the left A1 at 80 dB SPL. Aged A1 units displayed increased spontaneous (29%), peak (24%), and steady state response rates (38%) than did young A1 units. This was true across all A1 layers, although age-related differences were significantly greater for layers I-III (43% vs 18%) than lower layers. There was a significant age-related difference in the depth and duration of post-onset suppression between young and aged upper layer A1 units. The present functional differences across layers were consistent with studies showing greatest losses of gamma-aminobutyric acid (GABA) markers in superficial layers of A1 and with anatomic studies showing highest levels of inhibitory neurons located in superficial cortical layers. The present findings were also consistent with aging studies suggesting loss of functional inhibition in other cortical sensory systems., (Copyright 2009 Elsevier B.V. All rights reserved.)

Aims: The type 2 muscarinic receptor (M2R) differs from the other G-protein-coupled muscarinic receptor (type 4, or M4R) in tissue distribution and physiologic effects. We studied the impact of these receptors on sleep and arousal by using M2R and M4R knock-out (KO) mice., Main Methods: M2R and M4R KO and genetically intact mice were compared in terms of normal patterns of sleep, responses to sleep loss, infectious challenge and acoustic startle, and acoustic prepulse inhibition of startle (PPI)., Key Findings: Under basal conditions, M2R and M4R KO mice do not differ from the background strain or each other in the amount or diurnal pattern of sleep, locomotor activity, and body temperature. After enforced sleep loss, M2R KO mice, in contrast to the other two strains, show no rebound in slow-wave sleep (SWS) time, although their SWS is consolidated, and they show a greater rebound in time spent in REMS (rapid-eye-movement sleep) and REMS consolidation. During influenza infection, M2R KO mice, as compared with the other strains, show marked hypothermia and a less robust increase in SWS. During Candida albicans infection, M2R KO mice show a greater increase in SWS and a greater inflammatory response than do the other strains. M2R KO mice also show greater acoustic startle amplitude than does the background strain, although PPI was not different across the 3 strains over a range of stimulus intensities., Significance: Taken together, these findings support different roles for M2R and M4R in the modulation of sleep and arousal during homeostatic challenge., (Copyright 2009 Elsevier Inc. All rights reserved.)

Phenocopies may confound the clinical diagnoses of hereditary disorders. We report phenocopies in Multiple Endocrine Neoplasia type 1 (MEN1), an autosomal dominant disorder, characterised by the combined occurrence of parathyroid, pituitary and pancreatic tumours. We studied 261 affected individuals from 74 families referred with a clinical diagnosis of MEN1 and sought inconsistencies between the mutational and clinical data. We identified four patients from unrelated families with phenocopies. Patients 1 and 2 from families with MEN1, developed prolactinomas as the sole endocrinopathy but they did not harbour the germline MEN1 mutation present in their affected relatives. Patient 3, had acromegaly and recurrent hypercalcaemia following parathyroidectomy, whilst patient 4 had parathyroid tumours and a microprolactinoma. Patients 3 and 4 and their relatives did not have MEN1 mutations, but instead had familial hypocalciuric hypercalcaemia (FHH) due to a calcium-sensing receptor mutation (p.Arg680Cys), and the hyperparathyroidism-jaw tumour (HPT-JT) syndrome due to a hyperparathyroidism type 2 deletional-frameshift mutation (c.1239delA), respectively. Phenocopies may mimic MEN1 either by occurrence of a single sporadic endocrine tumour in a patient with familial MEN1, or occurrence of two endocrine abnormalities associated with different aetiologies. Phenocopies arose in >5% of MEN1 families, and awareness of them is important in the clinical management of MEN1 and other hereditary disorders.

Objective: Optimizing glycemic control in diabetic patients undergoing maintenance hemodialysis requires accurate assessment. We hypothesize that 1) 48-h continuous glucose monitoring (CGM) provides additional, clinically relevant, information to that provided by the A1C measurement and 2) glycemic profiles differ significantly between day on and day off dialysis., Research Design and Methods: With the use of GlucoDay S, 48-h CGM was performed in 19 type 2 diabetic subjects undergoing hemodialysis to capture consecutive 24-h periods on and off dialysis. Energy intake was calculated using food diaries. A1C was assayed by a high-performance liquid chromatography method., Results: CGM data were available for 17 subjects (13 male) with a mean (range) age of 61.5 years (42-79 years) and diabetes duration of 18.8 years (4-30 years). The 24-h CGM area under the glucose curve and 24-h mean glucose values were significantly higher during the day off dialysis than on dialysis (5,932.1 +/- 2,673.6 vs. 4,694 +/- 1,988.0 mmol x 3 min(-1) x l(-1), P = 0.022, and 12.6 +/- 5.6 vs. 9.8 +/- 3.8 mmol/l, P = 0.013, respectively), independent of energy intake. Asymptomatic hypoglycemia occurred in 4 subjects, 3 within 24 h of dialysis, and the glucose nadir in 14 subjects occurred within 24 h of dialysis., Conclusions: Glucose values are significantly lower on dialysis days than on nondialysis days despite similar energy intake. The risk of asymptomatic hypoglycemia was highest within 24 h of dialysis. Physicians caring for patients undergoing hemodialysis need to be aware of this phenomenon and consider enhanced glycemic monitoring after a hemodialysis session. CGM provides glycemic information in addition to A1C, which is potentially relevant to clinical management.

Neurophysiological studies of salicylate-induced tinnitus have generally been carried out under anesthesia, a condition that abolishes the perception of tinnitus and depresses neural activity. To overcome these limitations, measurement of salicylate induced tinnitus were obtained from rats using schedule induced polydipsia avoidance conditioning (SIPAC) and gap pre-pulse inhibition of acoustic startle (GPIAS). Both behavioral measures indicated that tinnitus was present after treatment with 150 and 250 mg/kg of salicylate; measurements with GPIAS indicated that the pitch of the tinnitus was near 16 kHz. Chronically implanted microwire electrode arrays were used to monitor the local field potentials and spontaneous discharge rate from multiunit clusters in the auditory cortex of awake rats before and after treatment with 150 mg/kg of salicylate. The amplitude of the local field potential elicited with 60 dB SPL tone bursts increased significantly 2h after salicylate treatment particularly at 16-20 kHz; frequencies associated with the tinnitus pitch. Field potential amplitudes had largely recovered 1-2 days post-salicylate when behavioral results showed that tinnitus was absent. The mean spontaneous spike recorded from the same multiunit cluster pre- and post-salicylate decreased from 22 spikes/s before treatment to 14 spikes/s 2h post-salicylate and recovered 1 day post-treatment. These preliminary physiology data suggest that salicylate induced tinnitus is associated with sound evoked hyperactivity in auditory cortex and spontaneous hypoactivity.

Environmental noise can alter endocrine, reproductive and cardiovascular function, disturb sleep/wake cycles, and can mask normal communication between animals. These outcomes indicate that noise in the animal facility might have wide-ranging affects on animals, making what laboratory animals hear of consequence for all those who use animals in research, not just the hearing researcher. Given the wide-ranging effects of noise on laboratory animals, routine monitoring of noise in animal facilities would provide important information on the nature and stability of the animal environment. This special issue will highlight the need for more thorough monitoring and will serve as an introduction to noise and its various effects on animals.

The fact that so little is currently known about the pathophysiology of tinnitus is no doubt partly due to the relatively slow development of an animal model. Not until the work of Jastreboff et al. (1988a, b) did tinnitus researchers have at their disposal a method of determining whether their animals experienced tinnitus. Since then, a variety of additional animal models have been developed. Each of these models will be summarized in this chapter. It is becoming increasingly clear that in order to study tinnitus effectively, researchers need some verification that a drug, noise exposure or other manipulation is causing tinnitus in their animals. As this review will highlight, researchers now have a variety of behavioral options available to them.

Objectives: Associations between calcium-sensing receptor (CaSR) polymorphisms and serum calcium, PTH and bone mineral density (BMD) have been reported by six studies. However, three other studies have failed to detect such associations. We therefore further investigated three CaSR coding region polymorphisms (Ala986Ser, Arg990Gly and Gln1011Glu) for associations with indices of calcium homeostasis and BMD and for alterations in receptor function., Patients and Design: One hundred and ten adult, Caucasian, female, dizygotic twin pairs were investigated for associations between the three CaSR polymorphisms and serum calcium, albumin, PTH, 25-hydroxyvitamin D(3) (25OHD(3)), 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)], urinary calcium excretion and BMD. Each polymorphic CaSR was also transfected into HEK293 cells and functionally evaluated., Results: There was a lack of association between each of these three CaSR polymorphisms and serum calcium corrected for albumin, PTH, 25OHD(3), 1,25(OH)(2)D(3), urinary calcium excretion or BMD at the hip, forearm and lumbar spine. These findings were supported by a lack of functional differences in the dose-response curves of the CaSR variants, with the EC(50) values (mean +/- SEM) of the wild-type (Ala986/Arg990/Gln1011), Ser986, Gly990 and Glu1011 CaSR variants being 2.74 +/- 0.29 mm, 3.09 +/- 0.34 mm (P > 0.4), 2.99 +/- 0.23 mm (P > 0.4) and 2.96 +/- 0.30 mm (P > 0.5), respectively., Conclusions: Our study, which was sufficiently powered to detect effects that would explain up to 5%, but not less than 1%, of the variance has revealed that the three CaSR polymorphisms of the coding region have no major influence on indices of calcium homeostasis in this female population, and that they do not alter receptor function.

Hearing in laboratory animals is a topic that traditionally has been the domain of the auditory researcher. However, hearing loss and exposure to various environmental sounds can lead to changes in multiple organ systems, making what laboratory animals hear of consequence for researchers beyond those solely interested in hearing. For example, several inbred mouse strains commonly used in biomedical research (e.g., C57BL/6, DBA/2, and BALB/c) experience a genetically determined, progressive hearing loss that can lead to secondary changes in systems ranging from brain neurochemistry to social behavior. Both researchers and laboratory animal facility personnel should be aware of both strain and species differences in hearing in order to minimize potentially confounding variables in their research and to aid in the interpretation of data. Independent of genetic differences, acoustic noise levels in laboratory animal facilities can have considerable effects on the inhabitants. A large body of literature describes the nonauditory impact of noise on the biology and behavior of various strains and species of laboratory animals. The broad systemic effects of noise exposure include changes in endocrine and cardiovascular function, sleep-wake cycle disturbances, seizure susceptibility, and an array of behavioral changes. These changes are determined partly by species and strain; partly by noise intensity level, duration, predictability, and other characteristics of the sound; and partly by animal history and exposure context. This article reviews some of the basic strain and species differences in hearing and outlines how the acoustic environment affects different mammals.