Exploring pain among young people who have completed treatment for acute lymphoblastic leukemia: experiences of youth and caregivers.

PURPOSE: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed among individuals <14 years of age. The disease and its treatments are associated with negative side effects, including pain, which is both prevalent and distressing. Little is known about pain experiences in this population, which has slowed efforts to identify strategies to mitigate and cope with this adverse effect. This study sought to explore youth's and their caregiver's experiences with, and perspectives of, pain in the context of pediatric cancer treatment. METHODS: Youth and one of their caregivers were recruited through (omitted for peer review). Following completion of a demographic survey, youth and one of their caregivers were interviewed separately using a semi-structured, one-on-one interview guide. Demographic information was analyzed with descriptive statistics, and interviews were transcribed verbatim and analyzed using reflexive thematic analysis. RESULTS: Youth (n = 19; Mage = 15.3 years) and caregiver (n = 19; Mage = 45.4 years) perspectives informed 4 themes: (1) my pain experience is nuanced, multidimensional, and is changing over time; (2) the cancer experience has changed the way I experience and respond to pain; (3) I used strategies to manage pain, and not all of them worked; and (4) my pain experience was influenced by people around me. CONCLUSIONS: Findings extend prior work, suggesting that pain is common, distressing, multidimensional, and influenced by social context. Results highlight the number of ways in which youth and their caregivers attempt to manage their pain and factors influencing pain experiences. Greater efforts are needed to address pain during cancer treatment and survivorship.

Endothelial regulation of calmodulin expression and eNOS-calmodulin interaction in vascular smooth muscle.

Calmodulin (CaM) is a Ca2+ sensor protein that is required for numerous vascular smooth muscle cell (VSMC) functions. Since CaM is not expressed enough for its many target proteins, factors that modulate its expression and interactions with targets in VSMCs can have extensive effects on vascular functions. VSMCs receive many regulatory inputs from endothelial cells (ECs). However, it is unknown if ECs regulate vascular functions via controlling expression of CaM and its interactions in VSMCs. In this work, we tested the hypothesis that ECs also affect VSMC signaling via regulation of CaM expression and interactions with its target proteins in VSMCs. Using ECs and VSMCs isolated from the same vessels and grown in a co-culture system, we observed that the presence of proliferating ECs significantly upregulates total CaM expression in VSMCs. An imaging module was devised to concurrently measure free Ca2+ and CaM levels in VSMCs in co-culture with ECs. Using indo-1/AM and a CaM biosensor built from a modified CaM-binding sequence of endothelial nitric oxide synthase (eNOS), this system revealed that in response to a generic Ca2+ signal, free Ca2+-bound CaM level is enhanced ~ threefold in VSMCs in co-culture with proliferating ECs. Interestingly, VSMCs express eNOS and eNOS-CaM association in response to the same Ca2+ stimulus is also enhanced ~ threefold in VSMCs co-cultured with ECs. Mechanistically, the endothelium-dependent upregulation of CaM in VSMCs is not affected by inhibition of NO production or endothelin receptors but is prevented by inhibition of vascular endothelial growth factor receptors. Consistently, VEGF-A level is upregulated in VSMCs co-cultured with proliferating ECs. These data indicate a new role of the endothelium in regulating vascular functions via upregulating CaM and its interactions in VSMCs.

First Report of Aphanomyces euteiches Race 1 and Race 2 Causing Aphanomyces Root Rot on Alfalfa (Medicago sativa) in South Dakota.

Aphanomyces euteiches causes Aphanomyces root rot (ARR) in alfalfa (Medicago sativa), along with root rot on many other legumes, including pea, clover, and lentil (Malvick et al., 2009). In 2020, South Dakota (SD) planted the most acres of alfalfa in the United States, which demonstrates the importance of alfalfa to the state. Several SD growers reported alfalfa establishment problems likely to be associated with ARR. Soil samples were collected from 16 fields under commercial alfalfa production in Lake County, SD in June 2020. Composite soil samples based on 24 subsamples were collected in a W-shaped pattern at a depth of 15 cm. Collected soil was sieved, and 80 cm3 was placed in plastic pots (6 cm x 6 cm). Each pot was planted with 25 seeds, covered with an additional 15 cm3 soil, and placed in a growth chamber with a 16-hour photoperiod at temperatures of 24 and 19 ℃ (day and night). Alfalfa seedlings, including Saranac (susceptible to races R1 and R2), WAPH-1 (resistant only to R1), WAPH-5 (resistant to both R1 and R2), and Mustang 625 (resistant to both R1 and R2 and coated with mefenoxam) grew in collected soil for 7 days, followed by 4 days under flooded conditions. Trays were drained, and at 21 days after planting (DAP), roots were removed from soil, washed in distilled water, and rated to measure severity of disease symptoms (Samac et al., 2015). The average severity index (ASI) used a 1-5 disease severity scale, 5 being a dead plant and 1 being no symptoms present (http://www.naaic.org/stdtests/Aphano.html). Race was based on ASI where R1 included an ASI of ≥3 for Saranac and <3 for WAPH-1, and R2 included an ASI of >3.0 for Saranac and WAPH-1 and <3.0 for WAPH-5 (Malvick and Grau, 2001). Race-typing experiments were repeated twice with six replicate pots per alfalfa cultivar per experiment and determined the presence of both R1 and R2 in Lake County, SD. ASI values for Mustang 625 and WAPH-5 were similar across all fields evaluated, which indicates limited confounding effects of other root rotting pathogens. DNA was extracted from three symptomatic roots from each field and was PCR amplified using A. euteiches specific primers (Vandemark et al., 2002). A PCR product was observed in all 16 fields evaluated, and the absence of a product was observed when DNA was extracted from alfalfa roots grown in vermiculite. Following race-typing, infected alfalfa roots were surfaced sterilized and placed on Aphanomyces selective media consisting of mefenoxam and benomyl in cornmeal agar (CMA) (Pfender et al., 1984). Isolates were identified as A. euteiches based on hyphal morphology (Malvick and Grau, 2001). Alfalfa seedlings (Saranac) were grown in vermiculite under growth conditions used for the race-typing assay and inoculated 6 DAP with two isolates of A. euteiches. Inoculation was completed using half plates of one week old A. euteiches mycelium on CMA blended with one liter of water (Samac et al., 2015). At 35 DAP, control alfalfa seedlings inoculated with blended CMA and water remained asymptomatic, and alfalfa infected with A. euteiches displayed symptoms including honey-brown colored lesions. For confirmation of Koch's postulates, DNA from three re-infected seedlings was again PCR amplified using A. euteiches specific primers and confirmed our previous work. This is the first report of either R1 or R2 of A. euteiches causing ARR on alfalfa in SD. To avoid future yield loss, SD growers should consider planting available alfalfa cultivars that have resistance to both races of A. euteiches.

First-in-Human Phase I Study of Merestinib, an Oral Multikinase Inhibitor, in Patients with Advanced Cancer.

BACKGROUND: The purpose of this nonrandomized, open-label, phase I study (NCT01285037) was to evaluate the safety and tolerability of merestinib, an oral antiproliferative and antiangiogenic kinase inhibitor, and to determine a recommended phase II dose and schedule for patients with advanced cancer. MATERIALS AND METHODS: This was a multicenter, nonrandomized, open-label, phase I study of oral merestinib consisting of six parts: dose escalation (part A), followed by a four-cohort dose-confirmation study (part B) and subsequently a four-part dose expansion and combination safety testing of merestinib with standard doses of cetuximab (part C), cisplatin (part D), gemcitabine and cisplatin (part E), and ramucirumab (part F) in patients with specific types of advanced cancers. Safety, tolerability, antitumor activity, and pharmacokinetics were evaluated in all cohorts. RESULTS: The dose escalation, confirmation, and expansion results support the dosing of merestinib at 120 mg once daily, based on acceptable exposure and safety at this dose. One complete response was observed in a patient with cholangiocarcinoma, and three patients with cholangiocarcinoma achieved a partial response. Overall, 60 (32%) of the 186 patients enrolled in the study had a best response of stable disease. CONCLUSION: This study demonstrates that merestinib has a tolerable safety profile and potential anticancer activity and warrants further clinical investigation. IMPLICATIONS FOR PRACTICE: Merestinib treatment in patients with advanced cancer demonstrated an acceptable safety profile and potential antitumor activity, supporting its future development in specific disease populations as a monotherapy and/or in combination with other therapies.

Suppression of store-operated Ca2+ entry by activation of GPER: contribution to a clamping effect on endothelial Ca2+ signaling.

The G protein-coupled estrogen receptor 1 (GPER, formerly also known as GPR30) modulates many Ca2+-dependent activities in endothelial cells. However, the underlying mechanisms are poorly understood. We recently reported that GPER acts to prolong cytoplasmic Ca2+ signals by interacting with and promoting inhibitory phosphorylation of the plasma membrane Ca2+-ATPase. In the present study, we examined the role of GPER activation in modulating store-operated Ca2+ entry (SOCE) via effects on the stromal interaction molecule 1 (STIM1). GPER activation by agonist G-1 reduces the peak but prolongs the plateau of bradykinin-induced Ca2+ signals in primary endothelial cells. G-1 dose-dependently inhibits thapsigargin-induced SOCE measured by the Mn2+ quenching method. GPER heterologous expression reduces SOCE, which is further pronounced by G-1 treatment. Consistently, GPER gene silencing in endothelial cells is associated with an increase in SOCE. Treatment with G-1 reduces puncta formation by STIM1 triggered by the activation of SOCE. The effect of GPER activation to inhibit SOCE is not affected by combined nonphosphorylatable substitutions at serines 486 and 668 on STIM1, but is substantially reduced by similar substitutions at serines 575, 608 and 621. Taken together with our recently reported inhibitory actions of GPER on Ca2+ efflux, the current data contribute to a model in which GPER acts to clamp agonist-induced cytoplasmic Ca2+ signals. Kinetic modeling based on current and reported data is used to estimate the overall effect of GPER activation on point activity of endothelial nitric oxide synthase during the time course of agonist-induced total Ca2+ signals.

Estrogen Enhances Linkage in the Vascular Endothelial Calmodulin Network via a Feedforward Mechanism at the G Protein-coupled Estrogen Receptor 1.

Estrogen exerts many effects on the vascular endothelium. Calmodulin (CaM) is the transducer of Ca(2+) signals and is a limiting factor in cardiovascular tissues. It is unknown whether and how estrogen modifies endothelial functions via the network of CaM-dependent proteins. Here we show that 17ß-estradiol (E2) up-regulates total CaM level in endothelial cells. Concurrent measurement of Ca(2+) and Ca(2+)-CaM indicated that E2 also increases free Ca(2+)-CaM. Pharmacological studies, gene silencing, and receptor expression-specific cell studies indicated that the G protein-coupled estrogen receptor 1 (GPER/GPR30) mediates these effects via transactivation of EGFR and subsequent MAPK activation. The outcomes were then examined on four distinct members of the intracellular CaM target network, including GPER/GPR30 itself and estrogen receptor α, the plasma membrane Ca(2+)-ATPase (PMCA), and endothelial nitric-oxide synthase (eNOS). E2 substantially increases CaM binding to estrogen receptor α and GPER/GPR30. Mutations that reduced CaM binding to GPER/GPR30 in separate binding domains do not affect GPER/GPR30-Gßγ preassociation but decrease GPER/GPR30-mediated ERK1/2 phosphorylation. E2 increases CaM-PMCA association, but the expected stimulation of Ca(2+) efflux is reversed by E2-stimulated tyrosine phosphorylation of PMCA. These effects sustain Ca(2+) signals and promote Ca(2+)-dependent CaM interactions with other CaM targets. Consequently, E2 doubles CaM-eNOS interaction and also promotes dual phosphorylation of eNOS at Ser-617 and Ser-1179. Calculations using in-cell and in vitro data revealed substantial individual and combined contribution of these effects to total eNOS activity. Taken together, E2 generates a feedforward loop via GPER/GPR30, which enhances Ca(2+)/CaM signals and functional linkage in the endothelial CaM target network.

#Boundaries: When patients become friends.

The personal use of social media by healthcare professionals is a hot topic within oncology practice settings. The widespread availability of both patient and provider personal information online threatens professional boundaries. This commentary article will provide an overview of the oncology nurses' responsibility to maintain boundaries and the potential risk to professional image if clients and families are able to access providers' personal information online. Work undertaken by the Advanced Practice Nursing Social Media Taskforce in the Hematology Oncology Transplant Program at the Alberta Children's Hospital will be presented including a literature review and development of a staff survey to explore perceptions and practices related to personal social media use by health professionals. Case scenarios of common social media challenges are explored and knowledge translation activities presented.

Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17ß-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities.

Making sense of the cancer journey: Pediatric cancer survivors' and their parents' autobiographical memories.

PURPOSE: Youth diagnosed with acute lymphoblastic leukemia (ALL) and their caregiver's experience a myriad of challenges in all domains of health that extend beyond treatment. Yet, little is known about how the cancer experience, and recollections associated with the experience, impact survivorship. We explored pediatric ALL survivors' and their caregivers' autobiographical memories of the cancer experience from diagnosis onwards. METHODS: Survivors of ALL, and one of their caregivers, were recruited through a local clinic. Survivors and their caregivers completed a demographic survey and semi-structured, private, one-on-one interviews. Demographic information were analyzed using descriptive statistics. Interviews were transcribed verbatim and analyzed using reflexive thematic analysis at the level of the individual and dyad. RESULTS: Insights from survivors (N = 19; Mage = 15.3 years) and their caregivers (n = 19; Mage = 45.4 years) were captured. Analyses generated two themes contingent on role (i.e., survivor or caregiver): (1) It is hard to recall my cancer experience and (2) We did as much as we could to manage our child's cancer experience and two unified themes (present in both survivors and their caregivers): (3) It took a village to get through the cancer experience and (4) The cancer diagnosis and experience has had a lasting impact. CONCLUSIONS: Findings highlight the varied and long-lasting ways cancer impacts survivors of pediatric ALL and their caregivers. Survivors had difficultly remembering their experience or felt that information was withheld and were acutely aware of their caregiver's distress. Caregivers were cautious and intentionally limited the information they shared. IMPLICATIONS FOR CANCER SURVIVORS: Survivors desired to be included within, or told about, decisions related to their healthcare and were acutely aware of their caregiver's distress. Efforts should be made to communicate with survivors (from diagnosis onward) openly and to consider strategies to minimize the short- and long-term impacts of pediatric ALL among survivors and their caregivers.

A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium.

AIMS: The Ca2+-binding protein calmodulin (CaM) modulates numerous target proteins but is produced insufficiently to bind all of them, generating a limiting CaM equilibrium. Menopause increases cardiac morbidity; however, it is unknown if the cardiac CaM equilibrium is affected by estrogen. We devised an assay to assess the effects of ovariectomy and estrogen treatment on the cardiac CaM equilibrium. MATERIALS AND METHODS: Sprague-Dawley rats received sham surgery or ovariectomy, followed by 2-week treatment with vehicle or 17ß-estradiol. Ca2+-saturated left ventricular (LV) lysates were processed through CaM sepharose columns, which retained CaM-binding proteins unoccupied by endogenous CaM. Eluants therefrom were subjected to a competitive binding assay against purified CaM and a CaM biosensor to assess the amounts of unoccupied CaM-binding sites. LV cellular composition was assessed by immunohistochemistry. KEY FINDINGS: LV eluants processed from sham animals reduce biosensor response by ~32%, indicating baseline presence of unoccupied CaM-binding sites and a limiting CaM equilibrium. Ovariectomy exacerbates the limiting CaM equilibrium, reducing biosensor response by ~65%. 17ß-estradiol treatment equalizes the difference between sham and ovariectomized animals. These changes reflect whole tissue responses and are not mirrored by changes in total surface areas of cardiomyocytes and fibroblasts. Consistently, Ca2+-dependent, but not Ca2+-independent, interaction between CaM and the cardiac inositol trisphosphate receptor (IP3R) is reduced following ovariectomy and is restored by subsequent 17ß-estradiol treatment. SIGNIFICANCE: Our assay provides a new parameter to assess tissue CaM equilibrium. The exacerbated limiting CaM equilibrium following estrogen loss may contribute to cardiac morbidity and is prevented by estrogen treatment.

Regulation of basal autophagy by calmodulin availability.

Macroautophagy (hereafter autophagy) is a process that degrades cellular components to maintain homeostasis. The Ca2+ sensor calmodulin (CaM) regulates numerous cell functions but is a limiting factor due to its insufficient availability for all target proteins. However, evidence that CaM availability regulates basal autophagy is lacking. Here, we have tested this hypothesis. CaM antagonists W-7, trifluoperazine and CGS9343b cause autophagosome accumulation and inhibit basal autophagic flux in the same manner as does chloroquine. These reagents promote the activity of AMP-activated protein kinase (AMPK) but not that of the mechanistic target of rapamycin (mTOR). Competitive binding assays using CaM sensors with different Ca2+ dependencies showed that chloroquine directly binds CaM in a Ca2+ -dependent fashion. The CaM antagonists have disparate effects on cytoplasmic Ca2+ , triggering from none to robust signals, indicating that their consistent inhibition of autophagy is due to inhibition of CaM and not Ca2+ . Chelating intracellular Ca2+ reduces the effect of the CaM antagonists to accumulate LC3-II, indicating that they do so by inhibiting CaM-dependent activities at basal Ca2+ level. The CaM antagonists cause lysosomal alkalinisation. Consistently, buffering CaM with a high-affinity CaM-binding protein that binds CaM at resting Ca2+ level increases lysosomal pH. Enhanced CaM buffering using a chimeric protein that contains two high-affinity CaM-binding sites that can collectively bind CaM at a large range of Ca2+ further increases lysosomal pH and increases LC3-II accumulation and AMPK activity, but not that of mTOR. These data demonstrate that CaM availability is required for basal autophagy.

Regulation of beta adrenoceptor-mediated myocardial contraction and calcium dynamics by the G protein-coupled estrogen receptor 1.

The G protein-coupled estrogen receptor 1 (GPER) produces cardioprotective effects. However, the underlying mechanisms are not well understood. We aimed to investigate the role of GPER in ß adrenoceptor-mediated cardiac contraction and myocardial signaling. In anesthetized animals, intrajugular administration of isoproterenol produces a rapid and sustained rise in left ventricular pressure (LVP) and increases ectopic contractions. Administration of the GPER agonist G-1 during the plateau phase of isoproterenol-induced LVP increase rapidly restores LVP to baseline levels and reduces the frequency of ectopic contractions. In freshly isolated cardiomyocytes, isoproterenol potentiates electrically induced peak currents of L-type Ca2+ channels (LTCC) and increases the potential sensitivity of their inactivation. Coadministration of G-1 prevents isoproterenol-induced potentiation of peak LTCC currents and makes channels more sensitive to being inactivated compared to isoproterenol alone. Isoproterenol treatment of cardiomyocytes without electrical stimulation triggers slow-rising Ca2+ signals that are inhibited by the ß1AR antagonist metoprolol but not by ß2AR antagonist ICI-118551. G-1 pretreatment dose-dependently suppresses isoproterenol-induced total Ca2+ signals and the amplitude and frequency of the intrinsic Ca2+ oscillatory deflections. Pretreatment with the GPER antagonist G-36 produces opposite effects, dose-dependently increasing these signals. ISO promotes robust phosphorylation of Cav1.2 channels at Ser1928. G-1 pretreatment inhibits isoproterenol-stimulated phosphorylation of Cav1.2 at Ser1928, while G-36 pretreatment enhances this signal. Our data indicate that GPER functions as an intrinsic component of ß1AR signaling to moderate myocardial Ca2+ dynamics and contraction.

Calcium/calmodulin regulates signaling at the α1A adrenoceptor.

Cardiovascular functions are mediated by multiple 7-pass transmembrane receptors whose activation promotes contraction or relaxation of the tissues. The α1 adrenoceptor type 1A plays important roles in the control of vascular tone and myocardial contractility via Ca2+-dependent actions. Here, using novel FRET-based biosensors, we identified a novel Ca2+-dependent interaction between calmodulin (CaM) and the human α1A adrenoceptor at the juxtamembranous region of its 4th submembrane domain (SMD4JM, a.a. 333-361). SMD4JM houses the known nuclear localization signal of α1A adrenoceptor (NLS, a.a. 334-349). We found that NLS itself also interacts with CaM, but with lower affinity and Ca2+ sensitivity, indicating that full interaction between CaM and α1A receptor in this region requires segment a.a. 333-361. Combined K353Q/L356A substitutions in the non-NLS segment of SMD4JM cause a 3.5-fold reduction in the affinity of CaM-SMD4JM interaction. Overexpression of wild-type α1A adrenoceptor in cells enhances phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) stimulated by A61603, while overexpression of the K353Q/L356A α1A receptor mutant significantly reduces this signal. Norepinephrine stimulates intracellular Ca2+ signals that are higher in cells overexpressing wild-type receptor but lower in cells overexpressing the K353Q/L356A receptor compared to non-transfected cells in the same microscopic environments. These data support a novel and important role for Ca2+-dependent CaM interaction at SMD4JM in α1A adrenoceptor-mediated signaling.

Human faecal collection methods demonstrate a bias in microbiome composition by cell wall structure.

Clinical trial faecal collections present challenges through geographical spread and inexperienced participants. Collection techniques have been developed and tested to overcome these challenges, but previous studies investigating these techniques have demonstrated a highly variable capacity for sample preservation. Furthermore, these studies typically only examine either preservation of genetic content or metabolites, not both. This study investigated the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for the preservation of both microbial DNA and microbial organic acid metabolites in human faecal samples when compared to frozen samples. Twenty six healthy adult participants were instructed to collect a bowel movement, subsample into collection tubes and immediately transfer the remaining bulk to -20 °C storage. Resulting organic acid concentrations remained comparable across methods when the preservation tubes were used correctly. The 16S rRNA gene sequencing data revealed twenty significantly different bacterial genera between the two collection methods. Ten Gram-negative genera were more abundant in the collection tubes, and ten Gram-positive genera were more abundant in the fresh frozen samples. This study has illustrated that faecal collection methods bias the microbial community profile according to Gram status and this should be considered when designing studies that collect and store human faecal samples.

Novel regulations of the angiotensin II receptor type 1 by calmodulin.

The angiotensin II receptor type 1 (AT1R) mediates many Ca2+-dependent actions of angiotensin II (AngII). Calmodulin (CaM) is a key transducer of Ca2+ signals in cells. Two locations on the receptor's submembrane domains (SMD) 3 and 4 are known to interact with CaM. However, the binding sites for CaM, biochemical properties of the interactions, and their functional impact are not fully understood. Using a FRET-based screening method, we identified a new binding site for CaM on SMD2 (a.a. 125-141), in addition to SMD3 and the juxtamembranous region of SMD4 (SMD4JM, a.a., 309-327). Simultaneous measurements of CaM binding and free Ca2+ show that the interactions are Ca2+-dependent, with disparate Kd and EC50(Ca2+) values within the physiological range of cytoplasmic Ca2+. Full interaction between CaM and SMD3 requires the entire domain (a.a. 215-242) and has an EC50(Ca2+) value in the range of resting cytoplasmic Ca2+, suggesting AT1R-CaM interaction can occur in resting conditions in cells. AngII induces robust ERK1/2 phosphorylation in primary vascular smooth muscle cells. This effect is suppressed by AT1R inhibitor losartan and virtually abolished by CaM antagonist W-7. AngII-induced ERK1/2 phosphorylation is suppressed in cells expressing mutant AT1R with reduced CaM binding at each identified binding domain. AngII triggers transient Ca2+ signals in cells expressing wild-type AT1R. These signals are reduced in cells expressing mutant AT1R with reduced CaM binding at SMD3 or SMD4JM, but are very slow-rising, low amplitude signal in cells expressing AT1R with reduced CaM binding at SMD2. The data indicate that CaM interactions with AT1R can occur at various domains, with different affinities, at different physiological Ca2+ levels, and are important for AT1R-mediated signaling.

A phase 1 study of the Janus kinase 2 (JAK2)V617F inhibitor, gandotinib (LY2784544), in patients with primary myelofibrosis, polycythemia vera, and essential thrombocythemia.

Mutations in Janus kinase 2 (JAK2) are implicated in the pathogenesis of Philadelphia-chromosome negative myeloproliferative neoplasms, including primary myelofibrosis, polycythemia vera, and essential thrombocythemia. Gandotinib (LY2784544), a potent inhibitor of JAK2 activity, shows increased potency for the JAK2V617F mutation. The study had a standard 3+3 dose-escalation design to define the maximum-tolerated dose. Primary objectives were to determine safety, tolerability, and recommended oral daily dose of gandotinib for patients with JAK2V617F-positive myelofibrosis, essential thrombocythemia, or polycythemia vera. Secondary objectives included estimating pharmacokinetic parameters and documenting evidence of efficacy by measuring clinical improvement. Thirty-eight patients were enrolled and treated (31 myelofibrosis, 6 polycythemia vera, 1 essential thrombocythemia). The maximum-tolerated dose of gandotinib was 120mg daily, based on dose-limiting toxicities of blood creatinine increase or hyperuricemia at higher doses. Maximum plasma concentration was reached 4h after single and multiple doses, and mean half-life on day 1 was approximately 6h. Most common treatment-emergent adverse events were diarrhea (55.3%) and nausea (42.1%), a majority of which were of grade 1 severity. Best response of clinical improvement was achieved by 29% of myelofibrosis patients. A ≥50% palpable spleen length reduction was observed at any time during therapy in 20/32 evaluable patients. Additionally, ≥50% reduction in the Total Symptom Myeloproliferative Neoplasm Symptom Assessment Form Score was seen in 11/21 (52%) and 6/14 patients (43%) receiving ≥120mg at 12 and 24 weeks respectively. Gandotinib demonstrated an acceptable safety and tolerability profile, and findings at the maximum-tolerated dose of 120mg supported further clinical testing. Clinicaltrials.gov identifier: NCT01134120.

Aging, peripheral nerve injury and nociception: effects of the antioxidant 16-desmethyltirilazad.

Peripheral neuropathies increase with aging, and reactive oxygen species contribute to the symptomatology of neuropathic pain disorders. In this study, we examined age-related differences in the therapeutic efficacy of pre- or post-treatments of the amino-steroidal antioxidant 16-desmethyltirilazad in delaying the onset and/or limiting the duration of tactile-evoked allodynia following the induction of peripheral mononeuropathies in rats. Two different models of nerve injury were utilized to induce allodynia in young and aged rats: (1) the chronic constriction injury (CCI) model of Bennett and Xie [Bennett GJ, Xie Y-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87-107]; (2) the partial sciatic nerve ligation (PSNL) model of Seltzer et al. [Seltzer Z, Dubner R, Shir YA. Novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43:205-18]. Calibrated von Frey filaments were used to examine changes in paw withdrawal threshold values. The results demonstrated that pre-treating young and aged rats with 16-desmethyltirilazad prior to the induction of peripheral mononeuropathies prevented the onset of neuropathic pain. However, once post-operative tactile allodynia was established, post-treatment therapy was ineffective at reversing the symptoms. These findings support the mediatory role of reactive oxygen species in neuropathic pain disorders, and suggest that the antiallodynic efficacy of antioxidant intervention is dependent on the time course of administration.

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic ß-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic ß-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

The effects of aging on thermal hyperalgesia and tactile-evoked allodynia using two models of peripheral mononeuropathy in the rat.

The effects of aging on the behavioral manifestations of neuropathic and inflammatory pain were investigated using two models of peripheral nerve injury. The left sciatic nerve of young and aged Fischer 344 FBNF1 hybrid rats (4-6 and 24-26 months old, respectively) was ligated using either the chronic constriction injury (CCI) model of Bennett and Xie or the partial sciatic nerve ligation (PSNL) model of Seltzer et al. A plantar analgesic meter was used to assess age-related differences in CCI- or PSNL-induced thermal hyperalgesia, and nerve injury-induced tactile-evoked allodynia was assessed with von Frey filaments. Aged animals subjected to the PSNL procedure developed a more vigorous and longer lasting thermal hyperalgesic response than did aged rats post-CCI. The CCI model incorporates a more prominent peripheral inflammatory component than the PSNL model. These data support the notion that the peripheral inflammatory response is diminished in aged rats.