M\u3csub\u3e1\u3c/sub\u3e Muscarinic Modulation of N-Type Calcium Channels: A Dissertation

The influx of calcium through N-type calcium channels (N-current) affects a myriad of neuronal functions. These include the triggering of synaptic release of neurotransmitter, adjustment of membrane potential and changes in gene transcription. N-channels are highly modulated proteins, so that N-current is attenuated or potentiated in response to environmental changes. In turn, the modulation of N-current has a direct effect on the downstream events, making the N-channel a focal point in neural signaling, and its modulation a mechanism for short term plasticity. The modulation of N-current by M1 muscarinic receptors (M1Rs) is of particular interest for several reasons. The M1R is instrumental in both cognition and memory formation as indicated by studies using either pharmacological agents aimed at M1Rs or knockout animals lacking M1Rs. Clinically, the M1R is an important target in the treatment of Alzheimer’s disease. Thus, like the N-channel, the M1R is an important element of neural signaling. Moreover, the stimulation of M1Rs affects N-current by through signaling pathways which despite being studied for decades, are not completely understood. For my dissertation I have investigated of M1R signaling on N-current using electrophysiological recordings of N-current from freshly dissociated neurons and from HEK cells expressing N-channels and M1Rs. Asking how one receptor affects one type of calcium channel would seem to be a simple question. However, the answer has many facets. Since M1Rs have multiple downstream effects and N-channels are highly modulated proteins, stimulation of M1Rs initiates several different pathways which modulate N-current. This thesis aims to unravel some of the complexities of the interactions of two vital components of neuronal signaling. Here I present the results of studies elucidating three different actions of M1signaling of N-current modulation. The first study I present here examines the effect of N-channel subunit composition on modulation of N-current. The stimulation of M1Rs in superior cervical ganglion (SCG) neurons elicits a distinct pattern of modulation; inhibiting N-current elicited by strong depolarizations and enhancing current elicited by lesser depolarizations. Thus M1Rs cause two simultaneous modulatory effects on N-current; increasing voltage sensitivity and decreasing overall conductance. I found the expression of the N-channel’s β subunit (CaVβ) determines the observed effect. Specifically when the isoform CaVβ2a is expressed M1 stimulation elicits enhancement without inhibition. Conversely, when CaVβ1b, CaVβ3, or CaVβ4 are expressed M1 stimulation elicits inhibition with out enhancement. These results fit a model in which both the enhancing and inhibiting effects of M1stimulation occur in all channels, but typically inhibition dominates. CaVβ2a blocks inhibition unmasking latent enhancement. Moreover, using mutants and chimeras I found palmitoylation of CaVβ2a at the N-terminus plays a key role in blocking inhibition. My findings predict the expression and localization of different CaVβ isoforms would dramatically alter modulation of N-current and thus may represent a previously unrecognized form of plasticity. The inhibition of N-current by M1Rs is controversial. It has been proposed recently that inhibition is directly attributable to the depletion of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during M1 stimulation. However, in our lab, we have found arachidonic acid (AA) release, which occurs subsequent to PtdIns(4,5)P2 hydrolysis, is both necessary and sufficient to elicit inhibition. Therefore, in a second study, I tested the effect of CaVβ expression on N-current during exogenous AA application and found a pattern of modulation identical to M1R stimulation. Furthermore, I took part in a collaborative project identifying the AA producing enzyme, diacylglycerol lipase (DAGL), to be a necessary component of the inhibitory pathway elicited by M1Rs. These findings provide increased evidence for AA release being a key factor in the M1R stimulated pathway of inhibition. Moreover, these discoveries identify the expression of CaVβ2a and use of specific DAGL inhibitors as a molecular and pharmacological strategy to block inhibition of N-current, respectively. These tools allow the dissection of downstream effects of M1R stimulation, so that other modulatory effects may be observed. The phosphorylation of N-channels by protein kinase C (PKC) blocks inhibition of current brought on by G-protein β and γ subunits (Gβγ) binding directly to the channel. Relief of Gβγ inhibition by other means has been identified as a mechanism of short term plasticity. M1Rs are known to simulate PKC, but a connection between M1Rs and PKC phosphorylation of Nchannels had not been demonstrated. I hypothesized that PKC stimulation may be occluded by other downstream effects of M1Rs. Therefore in a third study, I used a pharmacological approach on SCG neurons to dissect the PKC activating pathway from the other downstream effects of M1 stimulation. I observed modulation of N-current indicating a loss of Gβγ&#; inhibition, thus consistent with PKC phosphorylation of channels. This conclusion reveals another aspect of M1 modulation, which can function as a means of short term plasticity

The Ca2+ channel beta subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

In superior cervical ganglion (SCG) neurons, stimulation of M(1) receptors (M(1)Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M(1)Rs. In addition, techniques that diminish AA\u27s concentration during M(1)R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M(1)R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the beta subunit (Ca(V)beta) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M(1)Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca(V)2.2 and coexpressed with Ca(V)beta1b, Ca(V)beta3, or Ca(V)beta4 but enhanced activity of N channels containing Ca(V)beta2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca(V)beta2a, Ca(V)beta3, and Ca(V)beta4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca(V)beta with Ca(V)2.2. Further experiments with mutated, chimeric Ca(V)beta subunits and free palmitic acid revealed that palmitoylation of Ca(V)beta2a is essential for loss of inhibition. The data presented here fit a model in which Ca(V)beta2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca(V)beta2a toggles M(1)R- or NK-1R-mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M(1)Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca(V)beta2a

Potential impacts of climate change on agriculture and fisheries production in 72 tropical coastal communities

Climate change is expected to profoundly affect key food production sectors, including fisheries and agriculture. However, the potential impacts of climate change on these sectors are rarely considered jointly, especially below national scales, which can mask substantial variability in how communities will be affected. Here, we combine socioeconomic surveys of 3,008 households and intersectoral multi-model simulation outputs to conduct a sub-national analysis of the potential impacts of climate change on fisheries and agriculture in 72 coastal communities across five Indo-Pacific countries (Indonesia, Madagascar, Papua New Guinea, Philippines, and Tanzania). Our study reveals three key findings: First, overall potential losses to fisheries are higher than potential losses to agriculture. Second, while most locations (> 2/3) will experience potential losses to both fisheries and agriculture simultaneously, climate change mitigation could reduce the proportion of places facing that double burden. Third, potential impacts are more likely in communities with lower socioeconomic status

Seladelpar efficacy and safety at 3 months in patients with primary biliary cholangitis: ENHANCE, a phase 3, randomized, placebo-controlled study

Background and Aims: ENHANCE was a phase 3 study that evaluated efficacy and safety of seladelpar, a selective peroxisome proliferator-activated receptor-δ (PPAR) agonist, versus placebo in patients with primary biliary cholangitis with inadequate response or intolerance to ursodeoxycholic acid (UDCA). Approach and Results: Patients were randomized 1:1:1 to oral seladelpar 5 mg (n=89), 10 mg (n=89), placebo (n=87) daily (with UDCA, as appropriate). Primary end point was a composite biochemical response [alkaline phosphatase (ALP) < 1.67×upper limit of normal (ULN), ≥15% ALP decrease from baseline, and total bilirubin ≤ ULN] at month 12. Key secondary end points were ALP normalization at month 12 and change in pruritus numerical rating scale (NRS) at month 6 in patients with baseline score ≥4. Aminotransferases were assessed. ENHANCE was terminated early following an erroneous safety signal in a concurrent, NASH trial. While blinded, primary and secondary efficacy end points were amended to month 3. Significantly more patients receiving seladelpar met the primary end point (seladelpar 5 mg: 57.1%, 10 mg: 78.2%) versus placebo (12.5%) (p < 0.0001). ALP normalization occurred in 5.4% (p=0.08) and 27.3% (p < 0.0001) of patients receiving 5 and 10 mg seladelpar, respectively, versus 0% receiving placebo. Seladelpar 10 mg significantly reduced mean pruritus NRS versus placebo [10 mg: −3.14 (p=0.02); placebo: −1.55]. Alanine aminotransferase decreased significantly with seladelpar versus placebo [5 mg: 23.4% (p=0.0008); 10 mg: 16.7% (p=0.03); placebo: 4%]. There were no serious treatment-related adverse events. Conclusions: Patients with primary biliary cholangitis (PBC) with inadequate response or intolerance to UDCA who were treated with seladelpar 10 mg had significant improvements in liver biochemistry and pruritus. Seladelpar appeared safe and well tolerated

Seladelpar efficacy and safety at 3 months in patients with primary biliary cholangitis: ENHANCE, a phase 3, randomized, placebo-controlled study

Background and Aims: ENHANCEwas a phase 3 study that evaluated efficacy and safety of seladelpar, a selective peroxisome proliferator-activated receptor-delta (PPAR) agonist, versus placebo in patients with primary biliary cholangitis with inadequate response or intolerance to ursodeoxycholic acid (UDCA).Approach and Results: Patients were randomized 1:1:1 to oral seladelpar 5 mg (n= 89), 10 mg (n= 89), placebo (n= 87) daily (with UDCA, as appropriate). Primary end point was a composite biochemical response [alkaline phosphatase (ALP) < 1.67xupper limit of normal (ULN), >= 15% ALP decrease from baseline, and total bilirubin <= ULN] at month 12. Key secondary end points were ALP normalization at month 12 and change in pruritus numerical rating scale (NRS) at month 6 in patients with baseline score >= 4. Aminotransferases were assessed. ENHANCE was terminated early following an erroneous safety signal in a concurrent, NASH trial. While blinded, primary and secondary efficacy end points were amended to month 3. Significantly more patients receiving seladelpar met the primary end point (seladelpar 5 mg: 57.1%, 10mg: 78.2%) versus placebo (12.5%) (p < 0.0001). ALP normalization occurred in 5.4% (p= 0.08) and 27.3% (p < 0.0001) of patients receiving 5 and 10 mg seladelpar, respectively, versus 0% receiving placebo. Seladelpar 10 mg significantly reduced mean pruritus NRS versus placebo [10 mg: -3.14 (p= 0.02); placebo: -1.55]. Alanine aminotransferase decreased significantly with seladelpar versus placebo [5 mg: 23.4% (p= 0.0008); 10 mg: 16.7% (p= 0.03); placebo: 4%]. There were no serious treatment-related adverse events.Conclusions: Patients with primary biliary cholangitis (PBC) with inadequate response or intolerance to UDCA who were treated with seladelpar 10 mg had significant improvements in liver biochemistry and pruritus. Seladelpar appeared safe and well tolerated

STARD 2015: An Updated List of Essential Items for Reporting Diagnostic Accuracy Studies.

Incomplete reporting has been identified as a major source of avoidable waste in biomedical research. Essential information is often not provided in study reports, impeding the identification, critical appraisal, and replication of studies. To improve the quality of reporting of diagnostic accuracy studies, the Standards for Reporting of Diagnostic Accuracy Studies (STARD) statement was developed. Here we present STARD 2015, an updated list of 30 essential items that should be included in every report of a diagnostic accuracy study. This update incorporates recent evidence about sources of bias and variability in diagnostic accuracy and is intended to facilitate the use of STARD. As such, STARD 2015 may help to improve completeness and transparency in reporting of diagnostic accuracy studies

Working paper analysing the economic implications of the proposed 30% target for areal protection in the draft post-2020 Global Biodiversity Framewor

58 pages, 5 figures, 3 tables- The World Economic Forum now ranks biodiversity loss as a top-five risk to the global economy, and the draft post-2020 Global Biodiversity Framework proposes an expansion of conservation areas to 30% of the earth’s surface by 2030 (hereafter the “30% target”), using protected areas (PAs) and other effective area-based conservation measures (OECMs). - Two immediate concerns are how much a 30% target might cost and whether it will cause economic losses to the agriculture, forestry and fisheries sectors. - Conservation areas also generate economic benefits (e.g. revenue from nature tourism and ecosystem services), making PAs/Nature an economic sector in their own right. - If some economic sectors benefit but others experience a loss, high-level policy makers need to know the net impact on the wider economy, as well as on individual sectors. [...]A. Waldron, K. Nakamura, J. Sze, T. Vilela, A. Escobedo, P. Negret Torres, R. Button, K. Swinnerton, A. Toledo, P. Madgwick, N. Mukherjee were supported by National Geographic and the Resources Legacy Fund. V. Christensen was supported by NSERC Discovery Grant RGPIN-2019-04901. M. Coll and J. Steenbeek were supported by EU Horizon 2020 research and innovation programme under grant agreement No 817578 (TRIATLAS). D. Leclere was supported by TradeHub UKRI CGRF project. R. Heneghan was supported by Spanish Ministry of Science, Innovation and Universities, Acciones de Programacion Conjunta Internacional (PCIN-2017-115). M. di Marco was supported by MIUR Rita Levi Montalcini programme. A. Fernandez-Llamazares was supported by Academy of Finland (grant nr. 311176). S. Fujimori and T. Hawegawa were supported by The Environment Research and Technology Development Fund (2-2002) of the Environmental Restoration and Conservation Agency of Japan and the Sumitomo Foundation. V. Heikinheimo was supported by Kone Foundation, Social Media for Conservation project. K. Scherrer was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 682602. U. Rashid Sumaila acknowledges the OceanCanada Partnership, which funded by the Social Sciences and Humanities Research Council of Canada (SSHRC). T. Toivonen was supported by Osk. Huttunen Foundation & Clare Hall college, Cambridge. W. Wu was supported by The Environment Research and Technology Development Fund (2-2002) of the Environmental Restoration and Conservation Agency of Japan. Z. Yuchen was supported by a Ministry of Education of Singapore Research Scholarship Block (RSB) Research FellowshipPeer reviewe

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks