Journal articles on the topic 'Puketa Camp (N. Z.)'

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 μM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 μM isoproterenol or 100 μM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 μM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 μM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., β-agonists vs. nitric oxide).

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

We previously found that increased intravascular pressure decreased ischemic lung injury by a nitric oxide (NO)-dependent mechanism (Becker PM, Buchanan W, and Sylvester JT. J Appl Physiol 84: 803–808, 1998). To determine the role of cyclic nucleotides in this response, we measured the reflection coefficient for albumin (ςalb), fluid flux ( J˙), cGMP, and cAMP in ferret lungs subjected to either 45 min (“short”; n = 7) or 180 min (“long”) of ventilated ischemia. Long ischemic lungs had “low” (1–2 mmHg, n = 8) or “high” (7–8 mmHg, n = 6) vascular pressure. Other long low lungs were treated with the NO donor ( Z)-1-[ N-(3-ammoniopropyl)- N-( n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NONOate; 5 × 10−4 M, n = 6) or 8-bromo-cGMP (5 × 10−4 M, n = 6). Compared with short ischemia, long low ischemia decreased ςalb (0.23 ± 0.04 vs. 0.73 ± 0.08; P < 0.05) and increased J˙ (1.93 ± 0.26 vs. 0.58 ± 0.22 ml · min−1 · 100 g−1; P < 0.05). High pressure prevented these changes. Lung cGMP decreased by 66% in long compared with short ischemia. Lung cAMP did not change. PAPA-NONOate and 8-bromo-cGMP increased lung cGMP, but only 8-bromo-cGMP decreased permeability. These results suggest that ischemic vascular injury was, in part, mediated by a decrease in cGMP. Increased vascular pressure prevented injury by a cGMP-independent mechanism that could not be mimicked by administration of exogenous NO.

Abstract Objectives As individuals age, they become more susceptible to developing weight-related obesity comorbidities–which makes weight loss and weight maintenance key issues in older age groups. Prior research on the efficacy of weight loss programs across age groups has yielded inconsistent results. The Bright Line Eating (BLE) program, which follows a food addiction model that emphasizes abstinence from added sugars and processed flours, has been shown to be effective for weight loss and weight maintenance. This study builds on that research to assess age-related differences in the efficacy of the BLE program. Methods Participants in this study (n = 4509; 93.9% white; 95.6% female; 29.6% overweight, 58.2% obese at baseline) attended an 8-week BLE Boot Camp program and completed pre- and post-program surveys that measured demographic characteristics, anthropometrics, and psychosocial factors. We used two-way ANOVA to assess the effect of age on % weight loss from baseline (%WL) while accounting for race, gender, and program adherence. Logistic regression was used to estimate the effect of age on improvement in quality of life, energy level, and life satisfaction. Results Participants experienced 6.5%WL (SD = 5.2) with no significant difference across age groups (F = 1.5, P = 0.15). After completing the BLE Boot Camp program, 54.3% reported improved quality of life, 46.6% reported higher energy levels, and 60.4% described increased life satisfaction. Percent weight loss was associated with improvements in all of psychosocial factors that we examined (z = 13.8, P &lt; .000; z = 13.5, P &lt; .000; z = 12.4, P &lt; .000). Older study participants were more likely than younger to see an increase in energy level (z = 2.8, P = 0.01). Conclusions This evaluation of the BLE Boot Camp program demonstrated its success across all adult age groups. These results are particularly encouraging, given the need for feasible and scalable weight loss interventions that have been shown to be effective across all demographic groups. Funding Sources None to report.

Aims: The aim was to analyse the cost-effectiveness of an intensive weight-loss intervention for children compared with a low-intensity intervention. Methods: One hundred and fifteen overweight children (mean age 12.0 ± 0.4) were randomised to either the camp group (CG) ( N=59) or the standard group (SG) ( N=56). Participants in the CG were offered a six-week day-camp weight-loss programme followed by a family-based supportive programme containing four meetings during the succeeding 46 weeks. Participants in the SG were offered a weekly two-hour exercise session for six weeks. Changes in body mass index (BMI) and BMI z-score 12 months after inclusion were used to compare the effects of the two interventions. Incremental cost-effectiveness ratios (ICER) were estimated from the perspective of a Danish municipality. To achieve the required number of participants, an additional intervention was initiated one year later. Results: In comparison with the SG, the CG changed their mean BMI by −1.2 (95% CI −1.8 to −0.5). Compared with the SG children, the CG children changed their BMI z-score by −0.20 (95% CI −0.35 to −0.05). The ICER per decreased BMI point in the CG compared with the SG was DDK 24,928. Conclusions: Compared with the SG, the CG showed favourable effects after 12 months. However, the CG was more costly. The results observed in the present study may be helpful in guiding decision makers to take more informed decisions when choosing different types of intervention.

AbstractObjectiveEvaluate the feasibility, fidelity and preliminary efficacy of Camp NERF to prevent unhealthy weight gain and promote healthy behaviours in children during the summer.DesignCamp NERF was an 8-week, multicomponent, theory-based programme coupled with the US Department of Agriculture’s Summer Food Service Program. Twelve eligible elementary-school sites were randomized to one of three treatment groups: (i) Active Control (non-nutrition, -physical activity (PA), -mental health); (ii) Standard Care (nutrition and PA); or (iii) Enhanced Care (nutrition and PA, plus cognitive behavioural techniques) programming. Efficacy was determined by assessing mean change by group in child outcomes using hierarchical linear regression models.SettingLow-income, urban neighbourhoods in Columbus, OH, USA.ParticipantsEconomically disadvantaged, racial minority children of elementary school age (kindergarten–5th grade).ResultsEighty-seven child–caregiver dyads consented; eighty-one completed pre- and post-intervention assessments resulting in a 93·10 % retention rate. Delivery of the intended lesson occurred 79–90 % of the time. Of the children, 56·98 % (n 49) were female; 89·53 % (n 77) were Black. Overall mean change in BMI Z-score from baseline to post-intervention was −0·03 (se 0·05); change in BMI Z-score did not differ significantly between treatment group. Change in nutrition, PA, mental health or psychosocial outcomes did not differ between groups.ConclusionsResults from the current study demonstrate feasibility and fidelity, yet no intervention effect of Camp NERF. Instead, findings suggest that participation in structured programming of any type (health behaviour-related or not) may prevent unhealthy summer weight gain. Additional studies are needed to confirm findings. Results have implications for child nutrition policy addressing the issue of summer health.

Previous studies demonstrated inhibitory effects of nitric oxide (NO) and cGMP on ovarian steroidogenesis. This study examined the effects of NO on estrogen levels and cAMP accumulation from immature cultured rat granulosa cells. Granulosa cells were incubated with media alone (control), FSH or FSH plus increasing concentrations of the NO generator, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO). While FSH increased estrogen levels 15-fold compared with controls, DETA/NO inhibited FSH-stimulated aromatase activity in a dose-dependent manner. Time-course studies revealed that the inhibitory effects of DETA/NO on aromatase activity persisted throughout the 72 h culture period. Treatment with DETA/NO also inhibited the stimulatory effects of forskolin on estrogen production, indicating that NO can influence steroidogenesis by actions downstream of the FSH receptor. Incubation of cells with FSH plus DETA/NO increased cGMP accumulation over 100-fold, compared with cells treated with media or FSH alone. In this regard, a cGMP analog mimicked the inhibitory effects of NO on FSH- and forskolin-stimulated estrogen production, indicating a potential mechanism of NO action. NO also decreased FSH-stimulated (cAMP) accumulation from cultured cells, indicating an antagonistic effect of NO on the second messenger mediating FSH actions. These findings demonstrate that NO inhibits estrogen production from rat granulosa cells, potentially reflecting actions on the second messengers cGMP and cAMP.

In a newly characterized cultured porcine pulmonary artery (PA) preparation, 24-h treatment with the nitric oxide (NO) donor ( Z)-1-[ N-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) decreased the response to acutely applied DETA-NO compared with 24-h control (−log EC50 6.55 ± 0.12 and 5.02 ± 0.21, respectively). Treatment of PA with the cell-permeable superoxide dismutase mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride, did not change NO responsiveness in either freshly prepared or 24-h DETA-NO-treated PA. cGMP and cAMP phosphodiesterase activities were approximately equal in PA. Twenty-four-hour DETA-NO treatment did not change either cGMP or cAMP phosphodiesterase activities. Twenty-four hours in culture had no significant effect on soluble guanylyl cyclase (sGC) subunit mRNA expression, but 24-h DETA-NO treatment significantly decreased the expression of both sGCα1 and sGCβ1. sGCβ1 protein expression was 42 ± 4 ng/mg soluble protein. Twenty-four hours in culture without and with DETA-NO reduced sGCβ1 protein expression (36 ± 3 and 31 ± 3 ng/mg soluble protein, respectively, P < 0.025). Basal tissue cGMP [(cGMP)i] was significantly increased, and NO-induced (cGMP)i was significantly decreased by 24-h DETA-NO treatment. (cGMP)i normalized to the amount of sGC protein expressed in PA was significantly lower in PA treated for 24 h with DETA-NO compared with both freshly isolated and 24-h cultured PA. We conclude that prolonged NO treatment induces decreased acute NO responsiveness in part by decreasing both sGC expression and sGC-specific activity.

Regulation of L-type Ca2+ channels of vascular smooth muscle (VSM) cells by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent phosphorylation, which requires Mg2+ATP as a phosphate donor, has been reported (T. Ishikawa, J. R. Hume, and K. D. Keef. Circ. Res. 73: 1128-1137, 1993; Z. Xiong, N. Sperelakis, and C. Fenoglio-Preiser. J. Vasc. Res. 31: 271-279, 1994), and regulation by ATP has been demonstrated (Y. Ohya and N. Sperelakis. Circ. Res. 64: 145-154, 1989). However, it has not been elucidated whether the regulation by ATP is mediated by a mechanism that is distinct from phosphorylation. In the present study, we examined the effects of intracellularly perfused ATP on Ca2+ channel currents of VSM cells isolated from rat mesenteric arteries using a whole cell voltage clamp combined with an intracellular perfusion technique. Ba2+ currents (I(Ba)) through Ca2+ channels were evoked by depolarizing pulses from a holding potential of -80 mV with 130 mM Cs+ in the pipette and 100 mM Ba2+ in the bath. The decrease in the ATP concentration (from 5 to 0.1 mM) in the pipette caused a 45 +/- 5% (n = 8) reduction of maximal I(Ba) obtained at +40 mV within 10 min. The dose-response relation between I(Ba) and ATP showed a dissociation constant of 0.53 mM ATP. This concentration is much higher than that usually required for phosphorylation (e.g., few micromolar). Increase in the ATP (from 0.1 to 5 mM) caused an enhancement of maximal I(Ba) by 57 +/- 10% (n = 6), and this enhancement was not prevented in the presence of 30 microM H-7, a nonspecific inhibitor of protein kinases, or 1 microM protein kinase inhibitor, an inhibitor protein of cAMP-dependent protein kinase. These results indicate that slow Ca2+ channels in VSM cells are regulated by intracellular ATP, independently of phosphorylation, implying a direct regulatory action, such as a requirement for ATP binding to the inner surface of the channel, to exhibit activity.

Erdemli, G., Y. Z. Xu, and K. Krnjević. Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J. Neurophysiol. 80: 2378–2390, 1998. In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33°C, we studied the effects of brief hypoxia (2–3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid, and in the presence of kynurenate and bicuculline (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 ± 0.5 (SE) mV and their input resistance ( R in) fell by 23 ± 2.7%; in 30 cells under voltage clamp, membrane current increased by 114 ± 22.3 pA and input conductance ( G in) by 4.9 ± 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with “sharp” electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1–5 mM) or bath-applied glyburide (10 μM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (1.1 and 11 mM) and especially 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (11–33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The β-adrenergic agonist isoprenaline (10 μM) reduced hypoxic hyperpolarizations and decreases in R in ( n = 4) (and in another 11 cells corresponding changes in G in); and comparable but more variable effects were produced by internally applied 3′:5′-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP ( n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5′- O-(3-thiotriphosphate) (0.1–0.3 mM, n = 23) and especially guanosine 5′- O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-( p-sulfophenyl)theophylline (10–50 μM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of γ-aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein-dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.

Background and Introduction: An increasing number of deaths have accused at Tharparkar desert region of Sindh. Hence, a nutrition relief camp was set up at two remote villages named Haryar and Bhorilo. Visiting team consisted of nutritionists, dietitians, pediatricians and logistic support providers. Objective: To determine the prevalence of malnutrition among children (0-10 yrs) and its association with their living conditions. Methods: A cross sectional survey was conducted. Mothers with children aged 1 month to 10 years were invited to visit the camp.200 children were screened for malnutrition using anthropometric measurements including height, weight and Mid Upper Arm Circumference (MUAC). Dietary intake data was collected from mother including information about feeding practices, vaccination, disease history, and living conditions. Data was analyzed through SPSS 17. Inc using descriptive statistics. Results: Out of 200 children, 191 questionnaires were completed. Mean age was 3.6 ± 2.8 SD years. There were 101(52.9%) males and 90(47%) females. Results showed that 42.4 % (n=81) were affected by severe malnutrition (lower than 3rd percentile). Children less than 5 years were severely wasted as per WHO diagnostic criteria for Severe Acute Malnutrition (SAM) as indicated by 79(51%) children had a z score of -4 SD (for height & weight). Further analysis of children under 5 years into the mild acute (≤13.5 cm), moderate Acute (11.5-12.5 cm) and severe acute malnutrition (<11.5 cm) was carried out. The data demonstrated that 33.7 % (n=32) children had mild malnutrition, 37.7 % (n=58) suffered from moderate malnutrition and 15 % (n=23) had severe malnutrition. Food intake includes roti (Pakistani bread) and chatni (green chilies paste). There was no consumption of fruit, vegetable and milk due to no availability. Infants were breast fed and vaccinated but inappropriate and delayed weaning practices were reported by the mothers. Nearest medical facility/hospital was located at a distance of 15-30 miles. Conclusion: Major non nutrition related factors found to be contributing towards malnutrition were lack of education, water scarcity, non-availability of food, drinking water and lack of basic health facilities. There should be a provision of basic health facilities at community level. Health education and nutrition counseling should be included in their basic health initiatives.

Purpose: The study investigated the status of bone mineral density (BMD), serum Vitamin D and calcium and their association with performance outcomes in national-level Kho-Kho players. Methods: Kho-Kho players (n = 52; aged 16–31 years) undergoing training in a national camp were recruited. A lumbar spine (L1–L4) scan was performed using dual-energy X-ray absorptiometry to assess BMD. Blood samples were obtained to assess serum calcium and Vitamin D3 status. The 30-m sprint and Illinois tests were used to estimate speed and agility performance, respectively. The Spearman correlation and linear regression were used to ascertain the association between BMD and performance outcomes. Results: T-score was lower in 22.5% (mean ± standard deviation: 0.32 ± 0.96; range: −2 to +3), and serum Vitamin D3 was lower (<20 ng/ml) in 13.7% and insufficient (>20=<30 ng/ml) in 54.9% of players. No significant correlation between BMD (T-score and Z-score), serum Vitamin D3 and calcium was observed. T-score (r = 0.295, P < 0.05) and Z-score (r = 0.321, P < 0.05) were significantly positively correlated with speed but not with agility. An association was also found between Z-score (standardised-β: 1.38, confidence interval [CI]: 0.34–2.44, P < 0.01) and T-score (standardised-β: 1.49, CI: 0.37–2.62, P < 0.01) and speed. Conclusion: Low BMD and Vitamin D3 deficiency were prevalent amongst Kho-Kho players. Speed performance has an association with BMD.

IntroductionWars, such as in Ukraine, as well as terror attacks cause blast and ballistic casualties that are unusual in civilian practice. The immediate surgical response has not been systematically characterised due to the unpredictable, stochastic nature of such mass casualty events. We studied how the military trauma system managed civilian casualties during the Afghanistan conflict (2009-2014), to quantify and assess the surgical response to blast and ballistic injuries managed in a developed world paradigm and may be a tool for Emergency Preparedness, Resilience and Response (EPRR) planning.MethodThroughout the Afghanistan conflict (2009–2014) data was gathered prospectively by surgical teams at the UK-led military Medical Treatment Facility, Camp Bastion, Afghanistan. A total of 10,891 consecutive surgical cases were catalogued. Civilian cases were identified that had primary trauma surgery for blast and ballistic injuries and these formed the focus of this study (n=983). Statistical analyses with z-tests, significance was accepted at the 5% level (p<0·05).ResultsPaediatric wounding patterns when compared to adults show that children suffered consistently significantly more head (36·9% ;29·4%) and abdominal trauma (52·5%; 60·3%) for blast and ballistic injuries respectively (blast: 26·5%, z-2·263; p<0·05; 43·6%, z=-1·758, p<0·05; ballistic: 11·8%, z=2·926, p<0·01; 8%, z=-4·992; p<0·001; 60·3%, z=2·071, p<0·05). The three most common surgical procedures for blast injuries were debridement, amputation and laparotomy. For ballistic injuries, these were debridement, laparotomy and vascular procedures. A scalable predictive model of injury burden for blast and ballistic injuries in both adults and children is presented.ConclusionThis evidenced based, predictive model of the immediate response required to manage civilians injured by blast and ballistic mechanism can be applied to the conflict in Ukraine and to prepare for terror attacks on mass urban populations where blast and ballistic injuries are commonplace.

Human made conflicts and natural disasters led to a doubling of forced displacement in the past 10 years (United Nations High Commissioner for Refugees, 2022). Forcibly displaced people are generally challenged with severe mental and physical strains before, during and after their flight. Whereas forcibly displaced people show high levels of resilience (Dangmann et al., 2021), their risk of mental health struggles is markedly increased (Mesa-Vieira et al., 2022). The objective of this study was to explore the perception of regular participation in a co-designed and tailored sport and exercise intervention among adults living in a Greek refugee camp. The methodological approach relied on ethnography including informal discussions and participative observations. Data was collected and documented daily in a research diary over a period of eight months. Additionally, debrief meetings with the coaches (4 women and 4 men) were held regularly. These insights built the basis for a critical understanding and interpretation of 13 focus group discussions (n = 49, 19 women) and 38 self-recorded testimonies (17 women). Focus group discussions were conducted after a 10-week physical activity intervention period. The same participants were asked, after a 10-week follow-up, to provide a self-recorded testimony. Overall, 58 (55.8%) of the participants took part in the physical activities on a regular basis. Regular participation benefitted domains of psychological well-being, social cohesion, personal competencies, change in behavior and physiological health. However, some of the same domains were likewise negatively affected for certain participants, namely social cohesion and physiological health. The results indicate that organized sport and exercise activities have the potential to positively impact the living conditions of adults living in a Greek refugee camp. However, participation is not imperatively beneficial and only a specific target population can be addressed. References Dangmann, C., Solberg, Ø., Myhrene Steffenak, A. K., Høye, S., & Andersen, P. N. (2021). Syrian refugee youth resettled in Norway: Mechanisms of resilience influencing health-related quality of life and mental distress. Frontiers in Public Health, 9, Article 711451. https://doi.org/10.3389/fpubh.2021.711451 Mesa-Vieira, C., Haas, A. D., Buitrago-Garcia, D., Roa-Diaz, Z. M., Minder, B., Gamba, M., Salvador, D., Gomez, D., Lewis, M., Gonzalez-Jaramillo, W. C., Pahud de Mortanges, A., Buttia, C., Muka, T., Trujillo, N., & Franco, O. H. (2022). Mental health of migrants with pre-migration exposure to armed conflict: A systematic review and meta-analysis. The Lancet Public Health, 7(5), e469-e481. https://doi:10.1016/S2468-2667(22)00061-5 United Nations High Commissioner for Refugees. (2022). Global Trends. Forced Displacement in 2021. Retrieved from https://www.unhcr.org/publications/brochures/62a9d1494/global-trends-report-2021.html

NO and cGMP have antigrowth and anti-inflammatory effects on the vessel wall in response to injury. It is well established that after vascular injury proinflammatory cytokines are involved in vascular wall remodeling. The purpose of this study was to ascertain the signaling mechanisms involved in cGMP-dependent protein kinase (PKG) suppression by inflammatory cytokines in primary bovine aortic vascular smooth muscle cells (VSMC). Interleukin (IL)-Iβ, tumor necrosis factor (TNF)-α, and LPS decreased the mRNA and protein levels of PKG in VSMC. IL-Iβ, TNF-α, and LPS increased inducible nitric oxide synthase (iNOS) expression and cGMP production. Treatment of cells with selective inhibitors of iNOS or soluble guanylate cyclase (sGC) reversed the downregulation of PKG expression induced by cytokines and LPS. The NO donor ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) and 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), a NO-independent sGC activator, decreased PKG mRNA and protein expression in bovine aortic VSMC. Cyclic nucleotide analogs [8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP) and 8-(4-chlorophenylthio)adenosine 3,5′-cyclic monophosphate (CPT-cAMP)] also suppressed PKG mRNA and protein expression. However, CPT-cAMP was more effective than CPT-cGMP in decreasing PKG mRNA levels. Selective inhibition of PKA with the Rp isomer of 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphorothioate (Rp-8p-CPT cAMPS) prevented the downregulation of PKG by LPS. In contrast, the Rp isomer of 8-(4-chlorophenylthio)guanosine 3,5′-cyclic monophosphorothioate (Rp-8p-CPT cGMPS; inhibitor of PKG) had no effect on LPS-induced inhibition of PKG mRNA and protein expression. These studies suggest that cross-activation of PKA in response to iNOS expression by inflammatory mediators downregulates PKG expression in bovine aortic VSMC.

ABSTRACT The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism.

Significant language development differences occur in preschoolers, resolving these differences is a key objective of preschool education because young children's language abilities at the start of school, particularly their vocabulary abilities, are a strong predictor of later academic ability. Storytelling is one of the methods used to teach English, it makes the learning environment more interesting, energetic, and conversational. This study aims to examine storytelling in increasing children's vocabulary. The research method is a case study that uses observation, interviews, and documentation as data collection tools. The participants are an English teacher and seven children aged 4-5 years. Although, many early childhoods English teachers state that one of the weaknesses of children in learning English is a lack of vocabulary mastery because it can hamper the learning process. The findings show that storytelling is effective in increasing children's vocabulary. Vocabulary is the foundation for improving children's ability to speak English, both orally and in writing. Second language vocabulary is important to learn and develop for the quality of early childhood learners in language education. Keywords: early childhood, vocabulary, storytelling References: Agosto, D. E. (2016). Why Storytelling Matters: Unveiling the Literacy Benefits of Storytelling. Children and Libraries, 14(2), 21. https://doi.org/10.5860/cal.14n2.21 Chubb, J., Missaoui, S., Concannon, S., Maloney, L., & Walker, J. A. (2022). Interactive Storytelling for Children: A Case-Study of Design and Development Considerations for Ethical Conversational AI. Int. J. Child-Comp. Interact., 32(C). https://doi.org/10.1016/j.ijcci.2021.100403 Dewi, E. N. F., Hasanah, N., & Nurul, M. F. (2022). Enhancing Students’ Vocabulary Through Story Telling. EDULEC JOURNAL: Education, Language, and Culture. Egan, K. (1989). Teaching as Story Telling: An Alternative Approach to Teaching and Curriculum in the Elementary School. University of Chicago Press. https://books.google.com.mt/books?id=zNdLGAPzQT8C Ekawati, A. D. (2022). The Implementation of Total Physical Response (TPR) to Improve Student’s English Vocabulary During Pandemic. English Journal, 16(1). Fadli, M. R. (2021). Understand the design of qualitative research methods. humanics, [Memahami desain metode penelitian kualitatif]. Humanika, 21(1). Isik, M. A. (2016). The Impact of Storytelling on Young Ages. European Journal of Language and Literature Studies Articles, 2, 3. Kaur, A., Young, D., & Kirkpatrick, R. (2016). English Education Policy in Thailand: Why the Poor Results? In R. Kirkpatrick (Ed.), English Language Education Policy in Asia (Vol. 11, pp. 345–361). Springer International Publishing. https://doi.org/10.1007/978-3-319-22464-0_16 Khudhair, N. K., & Alnoori, B. S. M. (2017). Investigating EFL Preparatory School Teachers’ Perceptions Toward Using Storytelling Technique. Route Educational and Social Science Journal, 4(6). Kristiawan, D., Ferdiansyah, S., & Picard, M. (2022). Promoting Vocabulary Building, Learning Motivation, and Cultural Identity Representation through Digital Storytelling for Young Indonesian Learners of English as a Foreign Language. Ling, N. S., & Abdul Aziz, A. (2022). The Effectiveness of Game-based Learning Strategies on Primary ESL Learners’ Vocabulary Learning. International Journal of Academic Research in Progressive Education and Development, 11(2), Pages 845-860. https://doi.org/10.6007/IJARPED/v11-i2/13266 Malik, H., Humaira, M. A., Komari, A. N., Fathurrochman, I., & Jayanto, I. (2021). Identification of barriers and challenges to teaching English at an early age in Indonesia: An international publication analysis study. Linguistics and Culture Review, 5(1), 217–229. https://doi.org/10.21744/lingcure.v5n1.1485 McKay, P., & Guse, J. (2007). Five-Minute Activities for Young Learners. Cambridge University Press. https://books.google.co.id/books?id=BWKXLgrSv6gC Mufida, A., & Abidin, M. R. (2021). Designing a Board Game as a Media for Learning English for Children Aged 6-10 Years [Perancangan Board Game Sebagai Media Pembelajaran Bahasa Inggris Anak Usia 6-10 Tahun]. Jurnal Barik, 2(3). https://ejournal.unesa.ac.id/index.php/JDKV/ Noom-ura, S. (2013). English-Teaching Problems in Thailand and Thai Teachers’ Professional Development Needs. English Language Teaching, 6(11), p139. https://doi.org/10.5539/elt.v6n11p139 Pertiwi, A. B., Rahmawati, A., & Hafidah, R. (2021). English Vocabulary Learning Methods in Early Childhood [Metode Pembelajaran Kosakata Bahasa Inggris Pada Anak Usia Dini]. Kumara Cendekia, 9(2), 95. https://doi.org/10.20961/kc.v9i2.49037 Rahiem, M. D. H. (2021). Storytelling in early childhood education: Time to go digital. International Journal of Child Care and Education Policy, 15(1), 4. https://doi.org/10.1186/s40723-021-00081-x Stargatt, J., Bhar, S., Bhowmik, J., & Al Mahmud, A. (2022). Digital Storytelling for Health-Related Outcomes in Older Adults: Systematic Review. J Med Internet Res, 24(1), e28113. https://doi.org/10.2196/28113 Sunyakul, N., & Teo, A. (2020). Primary School English Teachers’ Application of Knowledge/Skills from Boot Camp to Their Classroom Teaching Practices and Factors Hindering Their Application. 13(1). Tarigan, H. G. (1986). Menulis sebagai suatu keterampilan berbahasa. Angkasa. https://books.google.co.id/books?id=XXoBtwAACAAJ Thornbury, S. (2002). How to teach vocabulary. Pearson educational. https://books.google.co.id/books?id=5qLkoAEACAAJ Udaya, M. (2022). Using Semantic Maps as A Teaching Strategy for Vocabulary Development. European Journal of English Language Teaching, 6(5). https://doi.org/10.46827/ejel.v6i5.4095 Zu, Y., Cheng, Z., Sun, Q., & Zhao, H. (2021). On the Problems and Countermeasures in English Vocabulary Teaching in Junior Middle Schools. 568.

Augmented cGMP- and diminished cAMP-signaling within cardiomyocytes is characteristic for failing hearts. Cyclic nucleotide phosphodiesterases (PDEs) comprise a family of cyclic-nucleotide hydrolyzing enzymes, controlling cAMP and cGMP levels. Among them the PDE-2A isoform has the unique property to be stimulated by cGMP but primarily hydrolyzing cAMP. This appears to mediate a negative cross-talk between both signaling pathways. However, a potential role for PDE-2A in the failing heart has not been addressed yet. Here we show that PDE-2A protein levels were ∼2-fold higher in failing human hearts as well as in a large animal heart failure model from dog hearts subjected to rapid-pacing (n≥6, p<0.05). Intriguingly, PDE-2A protein levels were normal in hypertrophied hearts from patients with preserved cardiac function who underwent aortic valve replacement. Chronic beta-adrenergic stimulation by catecholamine infusions enhanced cAMP hydrolyzing activity of PDE-2A by four-fold (n≥6, p<0.05) in rat hearts in vivo and in isolated cardiomyocytes (measured by radioimmunoassay and FRET-based sensors, respectively) and correlated with blunted beta-adrenergic responsiveness. Consistent with this observation, overexpressed PDE-2A, which localized to the sarcomeric Z-line, blunted the rise in cAMP by 70% (n≥6, p<0.05) and abolished the positive inotropic effect after acute beta-adrenergic stimulation by 70% (n≥6, p<0.05) in isolated cardiomyocytes. Notably, those cardiomyocytes also showed marked protection from norepinephrine-induced hypertrophic responses, e. g. 40% less increase in cell surface area (n≥10, p<0.05). In summary, PDE-2A is markedly upregulated in human and experimental failing hearts. This may constitute an important defense mechanism during cardiac stress, by antagonizing the cAMP-mediated toxic effects. Thus, activating myocardial PDE-2A may represent a new intracellular anti-adrenergic therapeutic strategy in heart failure.

Abstract Background While current estimates suggest that up to three million additional surgical procedures are needed to meet the needs of forcibly displaced populations, literature on surgical care for refugee or forced migrant populations has often focused on acute phase and war-related trauma or violence with insufficient attention to non-war related pathologies. To our knowledge, no study has compared refugee versus host population utilization of surgical services in a refugee camp-based hospital over such an extended period of twenty years. The aim of this paper is to first describe the patterns of surgical care by comparing refugee and host population utilization of surgical services in Nyarugusu refugee camp between 2000 and 2020, then evaluate the impact of a large influx of refugees in 2015 on refugee and host population utilization. Methods The study was based on a retrospective review of surgical logbooks in Nyarugusu refugee camp (Kigoma, Tanzania) between 2000 and 2020. We utilized descriptive statistics and multiple group, interrupted time series methodology to assess baseline utilization of surgical services by a host population (Tanzanians) compared to refugees and trends in utilization before and after a large influx of Burundian refugees in 2015. Results A total of 10,489 operations were performed in Nyarugusu refugee camp between 2000 and 2020. Refugees underwent the majority of procedures in this dataset (n = 7,767, 74.0%) versus Tanzanians (n = 2,722, 26.0%). The number of surgeries increased over time for both groups. The top five procedures for both groups included caesarean section, bilateral tubal ligation, herniorrhaphy, exploratory laparotomy and hysterectomy. In our time series model, refugees had 3.21 times the number of surgeries per quarter at baseline when compared to Tanzanians. The large influx of Burundian refugees in 2015 impacted surgical output significantly with a 38% decrease (IRR = 0.62, 95% CI 0.46–0.84) in surgeries in the Tanzanian group and a non-significant 20% increase in the refugee group (IRR = 1.20, 95% CI 0.99–1.46). The IRR for the difference-in-difference (ratio of ratios of post versus pre-intervention slopes between refugees and Tanzanians) was 1.04 (95% CI 1.00–1.07), and this result was significant (p=0.028). Conclusions Surgical care in conflict and post-conflict settings is not limited to war or violence related trauma but instead includes a large burden of obstetrical and general surgical pathology. Host population utilization of surgical services in Nyarugusu camp accounted for over 25% of all surgeries performed, suggesting some host population benefit of the protracted refugee situation in western Tanzania. Host population utilization of surgical services was apparently different after a large influx of refugees from Burundi in 2015.

Abstract Purpose In adults, diets rich in protein seem beneficial in relation to satiety, weight loss, and weight management; however, studies investigating dietary protein and weight development in children are scarce and inconsistent. This nonrandomized controlled trial aimed to investigate the effect of a higher protein diet during lifestyle intervention on anthropometry and metabolic biomarkers in children with overweight and obesity. Methods Children (n:208) were recruited from two multicomponent lifestyle camps. One camp was assigned as the intervention group. In the intervention group, carbohydrates-rich foods at breakfast and two in-between-meals were replaced with protein-containing foods to increase the amount of protein from ~ 10–15 energy percent (E%) per day to ~ 25E% per day. Other components were similar between groups. Anthropometry and biochemical measurements were collected at baseline, 10 weeks (after camp) and 52 weeks. Results The intervention group had a non-significant improvement in BMI-SDS (− 0.07 SD (− 0.19; 0.05), p = 0.24) compared to the control group, but in general, there was no effect of a higher protein diet on anthropometry and metabolic biomarkers. Overall, 10 weeks at camp resulted in a more favorable body composition [− 6.50 kg (p < 0.00), − 0.58 BMI-SDS (p < 0.00), and − 5.92% body fat (p < 0.00)], and improved metabolic health, with most changes maintained at 52 weeks. Conclusion A higher protein diet had no significant effect on body composition and metabolic health; however, these lifestyle camps are an efficiatious treatment strategy for childhood obesity. Clinical trial registration: clinicaltrials.gov with ID: NCT04522921. Preregistered August 21st 2020.

Background: It is challenging to maintain effects of public health interventions. For residential health camps benefits often disappear as the child returns home. Furthermore, long-term effects are often not measured or reported. This paper presents the study protocol for an evaluation of an extended maintenance intervention offered to children who have completed a 10-week residential health camp at one of the five Danish Christmas Seal Houses (DCSH). The target group of DSCH is 7–14-year-olds with social, mental, and/or overweight issues and the overall aim of the camp is to increase life satisfaction and a healthy lifestyle. The primary aim of this study is to assess the effectiveness of the maintenance intervention on children's life satisfaction (primary outcome) and BMI Z-score (secondary outcome) 1 year after health camp.Methods: The extended maintenance intervention is developed by DCSH and delivered to each child and family individually by an intervention coordinator to help children maintain positive benefits of the health camp on life satisfaction and health behaviors after returning to their homes. Intervention activities target the child and the family. The effect will be tested in a quasi-experimental design: The intervention is offered to half of the children at one of the five DSCH (intervention group, N~144) while the other half and the children at the other four DSCH receive a standard maintenance intervention (control group, N~894). Children will complete questionnaires on life satisfaction measured by an adapted version of the Cantril ladder and height and weight prior to health camp, at the end of health camp, 3 months and 1 year after the end of health camp. To enable per protocol analysis and nuanced interpretation of effect estimates, we will monitor the implementation of the intervention by a process evaluation study among children, parents, and follow up coordinators using qualitative and quantitative methods.Discussion: We present a systematic approach to evaluating practice-based interventions in a research design. The study will provide new knowledge on the effectiveness of individualized maintenance interventions on long-term effects on life satisfaction and weight loss among children.Trial registration: Prospectively registered at Current Controlled Trials ISRCTN 13011465 https://www.isrctn.com/ISRCTN13011465

AbstractDopamine and cAMP regulated phosphoprotein 32 kDa (DARPP-32) also known as phosphoprotein phosphatase-1 regulatory subunit 1B and encoded by the PPP1R1B gene is an inhibitor of protein phosphatase-1 and protein kinase A. DARPP-32 is expressed in a wide range of epithelial cells and some solid tumours; however, its role in breast cancer is only partially defined. DARPP-32 expression was determined using immunohistochemistry in two independent cohorts of early stage invasive breast cancer patients (discovery n = 1352; validation n = 1655), and 112 HER2 positive breast cancer patients treated with trastuzumab and adjuvant chemotherapy. PPP1R1B mRNA expression was assessed in the METABRIC cohort (n = 1980), using artificial neural network analysis to identify associated genes. In the discovery cohort, low nuclear expression of DARPP-32 was significantly associated with shorter survival (P = 0.041), which was independent of other prognostic variables (P = 0.019). In the validation cohort, low cytoplasmic and nuclear expression was significantly associated with shorter survival (both P = 0.002), with cytoplasmic expression independent of other prognostic variables (P = 0.023). Stronger associations with survival in oestrogen receptor (ER) positive disease were observed. In patients treated with trastuzumab, low nuclear expression was significantly associated with adverse progression-free survival (P = 0.031). In the METABRIC cohort, low PPP1R1B expression was associated with shortened survival of ER positive patients. Expression of CDC42 and GRB7, amongst others, were associated with PPP1R1B expression. This data suggests a role for DARPP-32 as a prognostic marker with clinical utility in breast cancer.

AbstractFree-recall paradigms have greatly influenced our understanding of memory. The majority of this research involves laboratory-based events (e.g., word lists) that are studied and tested within minutes. This literature shows that adults recall events in a temporally organized way, with successive responses often coming from neighboring list positions (i.e., temporal clustering) and with enhanced memorability of items from the end of a list (i.e., recency). Temporal clustering effects are so robust that temporal organization is described as a fundamental memory property. Yet relatively little is known about the development of this temporal structure across childhood, and even less about children’s memory search for real-world events occurring over an extended period. In the present work, children (N = 144; 3 age groups: 4–5-year-olds, 6–7-year-olds, 8–10-year-olds) took part in a 5-day summer camp at a local zoo. The camp involved various dynamic events, including daily animal exhibit visits. On day 5, children were asked to recall all the animals they visited. We found that overall recall performance, in terms of number of animals recalled, improved steadily across childhood. Temporal organization and recency effects showed different developmental patterns. Temporal clustering was evident in the response sequences for all age groups and became progressively stronger across childhood. In contrast, the recency advantage, when characterized as a proportion of total responses, was stable across age groups. Thus, recall dynamics in early childhood parallel that seen in adulthood, with continued development of temporal organization across middle to late childhood.

AbstractClass B1 of G protein-coupled receptors (GPCRs) comprises 15 members activated by physiologically important peptide hormones. Among them, vasoactive intestinal polypeptide receptor 2 (VIP2R) is expressed in the central and peripheral nervous systems and involved in a number of pathophysiological conditions, including pulmonary arterial hypertension, autoimmune and psychiatric disorders, in which it is thus a valuable drug target. Here, we report the cryo-electron microscopy structure of the human VIP2R bound to its endogenous ligand PACAP27 and the stimulatory G protein. Different from all reported peptide-bound class B1 GPCR structures, the N-terminal α-helix of VIP2R adopts a unique conformation that deeply inserts into a cleft between PACAP27 and the extracellular loop 1, thereby stabilizing the peptide-receptor interface. Its truncation or extension significantly decreased VIP2R-mediated cAMP accumulation. Our results provide additional information on peptide recognition and receptor activation among class B1 GPCRs and may facilitate the design of better therapeutics.

Renin is essential for angiotensin I generation and blood pressure regulation. Renin is stored in dense core granules in juxtaglomerular (JG) cells. Renin release is highly regulated with only a small percentage (2 to 5%) of the total renin content being released after maximal stimulation with agonists that increase cAMP. In vivo, stimulation of renin release resulted in rare events of renin granule disappearance as shown by electron microscopy. We found that stimulated‐renin release is in part mediated by exocytic proteins present in the renin granules. However, real‐time visualization of renin granule exocytosis has not been done. We hypothesize that renin exocytosis from JG cells is due to controlled exocytic events, termed kiss‐and‐run exocytosis, where fusion of the granule with the plasma membrane is transient and granule integrity is maintained. To study this we generated a new construct in which full length mouse renin was tagged with yellow fluorescent protein in its Carboxyl‐terminus (Renin‐YFP). First, we packed the Renin‐YFP construct in adenoviruses (Ad‐Ren‐YFP) and characterized its proper expression and activity in Att20 cells, an endocrine pituitary cell line that does not express endogenous renin. By Western Blot, we observed a band at the expected molecular mass of renin‐YFP (70 kDa), detected with antibodies against YFP or renin (n=4). Then, we tested whether Ad‐Renin‐YFP retains its enzymatic activity (rate of angiotensin I production from angiotensinogen) in Att20 cells. Renin activity was only detected in Att20 cells transduced with renin‐YFP (n=3; p<0.01). To monitor exocytosis of renin‐YFP, we transduced primary cultures of mouse JG cells. After 24 hs, we transferred JG cells to a 37C chamber and imaged YFP‐containing granules by TIRF microscopy. TIRF allows imaging of granule movements and changes in fluorescence intensity within 250 nm of the plasma membrane. Under baseline conditions we observed an average of 16±4 granules per cell docked with the plasma membrane (n=12). Translational movement (X–Y planes) of docked granules was negligible and most granules remained docked during 20 minutes of imaging. TIRF events were quantified over 10 minutes by measuring: A) recruitment of new granules, B) full fusion followed by granule disappearance and C) rapid axial movement (Z‐axis) of docked granules (observed as rapid bursts of fluorescence followed by return to baseline intensity). In the absence of cAMP, the total number of events per cell was low and no full fusion events were detected (1.5 ±0.5 total events per cell, n=4). After stimulation with cAMP, the number and frequency of events increased 4 fold to 5.3±1.0 events per cell (n=8, p<0.05). Only one full fusion event was detected (n=8 cells, 152 granules imaged). Most of the events caused by cAMP (73.5%) were due to fluorescence bursts of docked granules and 36.5% of events were caused by recruitment of newcomer granules to the TIRF field. We conclude that in JG cells, full fusion of granules is not the main mechanism of renin exocytosis. The rapid bursts in fluorescence intensity of docked granules suggest that kiss‐and‐run is the main mechanism of stimulated‐renin exocytosis. This is the first time that a renin‐YFP construct was characterized and renin exocytosis is imaged in JG cells.Support or Funding Information1RO3DK 105300;SDG17070121

AbstractIndividuals were found to anonymously predict positive election outcomes for their preferred candidate. Yet, there is little scientific knowledge about election predictions made in the context of same-camp political communications (i.e., partisan communications) that are presumably meant to encourage other supporters. In five studies of low-information elections and a study of hypothetical U.S. elections (n = 1889), we found that people tended to communicate favorable forecasts to others sharing their view, compared to the neutral point and to the actual election outcomes. On the other hand, negative framing reduced the positivity of forecasts in these communications to the extent that it led most participants to predict an election loss. This occurred in response to a single addressee acting discordantly and even more strongly when the election results were phrased as a drop. When both positive and negative framing options were available, this still negativity affected participants’ predictions even though only a minority selected the negative framing option. Thus, people tend to make optimistic election predictions in partisan communications, but this pattern is easily manipulable given subtle changes in the forecasting prompt, either by negative framing or selectable positive and negative framing.

AbstractAcute kidney injury (AKI) is common in patients hospitalized for COVID-19, complicating their clinical course and contributing to worse outcomes. Animal studies show that adenosine, inosine and guanosine protect the kidney against some types of AKI. However, until now there was no evidence in patients supporting the possibility that abnormally low kidney levels of adenosine, inosine and guanosine contribute to AKI. Here, we addressed the question as to whether these renoprotective purines are altered in the urine of COVID-19 patients with AKI. Purines were measured by employing ultra-high-performance liquid chromatography-tandem mass spectrometry with stable-isotope-labeled internal standards for each purine of interest. Compared with COVID-19 patients without AKI (n = 23), COVID-19 patients with AKI (n = 20) had significantly lower urine levels of adenosine (P < 0.0001), inosine (P = 0.0008), and guanosine (P = 0.0008) (medians reduced by 85%, 48% and 61%, respectively) and lower levels (P = 0.0003; median reduced by 67%) of the 2nd messenger for A2A and A2B adenosine receptors, i.e., 3’,5’-cAMP. Moreover, in COVID-19 patients with AKI, urine levels of 8-aminoguanine (endogenous inhibitor of inosine and guanosine metabolism) were nearly abolished (P < 0.0001). In contrast, the “upstream” precursors of renoprotective purines, namely 5’-AMP and 5’-GMP, were not significantly altered in COVID-19 patients with AKI, suggesting defective conversion of these precursors by CD73 (converts 5’-AMP to adenosine and 5’-GMP to guanosine). These findings imply that an imbalance in renoprotective purines may contribute to AKI in COVID-19 patients and that pharmacotherapy targeted to restore levels of renoprotective purines may attenuate the risk of AKI in susceptible patients with COVID-19.

Background: Eating disorders are commonly diagnosed psychiatric disorders in adolescents. Because of the severity of disease and difficulty of treatment, means of prevention are important. Girls Inc. is a national non-profit organization that delivers evidence-based programming for people identifying as girls. Their four-week summer camp includes curriculum for building relationships, economic and media literacy, body image, and self-esteem. Here, the Girls Inc. curriculum “Redefining Beauty” is evaluated for efficacy of changing attitudes and behaviors correlated with development of eating disorders. Methods: Pre-and-post curriculum self-report surveys were administered to 11-14-year-olds (n=28) at the Girls Inc. summer camp. Group C1 consisted of 11-12 year-olds and C2 were 13-14 year-olds. All participants present for the camp the day of surveying were included in the study. Likert scales were used to assess body image and self -esteem. Collins Child Female Scale determined dissonance between current and ideal body image. Data was also collected regarding the use of dieting and exercise to lose weight. Results: No significant differences were found. In a Likert scale question on body satisfaction, there was no significant difference between pre-and post-curriculum answers (X2(t-1.7056, p=0.1036)). Body dissonance was measured to have no significant difference between pre-and post-curriculum answers (Paired t-test(t=0.8467, p=0.4077)). In a question asking if participants have been on a diet, 41.4% answered yes. This data was compared between two groups, and no significant difference was found (X2(z=0.1357, p=0.7125)) . Conclusion: This research can be used to aid Girls Inc. and similar programs in updating or creating eating disorder prevention curriculum. Because research has shown that increasing self-esteem and improving body image leads to decreased risk of developing eating disorders, it is important that curriculum aimed at preventing eating disorders is measured for its success in improving these factors.

Abstract Objectives Objectives were to examine subjective sleep quality and daytime sleepiness of the German ice hockey junior national team prior to the world championship to identify athletes of concern and areas of optimization with the intention of equally preventing injury and enhancing performance. Methods Twenty-one athletes (Mage = 18.5 ± 0.6 years, Mheight = 181.7 ± 4.3 cm, Mweight = 81.4 ± 7.1 kg), playing for national (n = 13) and international (n = 8) home clubs, answered the Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale (ESS) before training camp (T1, day 1) and prior to tournament (T2, day 11). Results Overall, 9 players at T1 and 7 at T2 were identified as bad sleepers (PSQI > 5), while high sleepiness (ESS > 10) was found for 6 athletes at each measurement time. Group means and standard deviations reduced descriptively for PSQI (T1 = 5.38 ± 2.31, T2 = 4.57 ± 2.36) and ESS (T1 = 9.24 ± 3.74, T2 = 8.48 ± 3.28). Tendential differences were visible for PSQI in international-based players (Z = −1.7, p = 0.09) and ESS in first-national-league players (Z = −1.73, p = 0.08) over time. Higher PSQI values for international-based players (6.25 ± 2.6) were found compared to first-national-league (5.83 ± 1.60) and lower-league players (4.00 ± 2.08), with large effect sizes for lower-league compared to international (d = 0.95) and national players (d = 0.98) at T1 and small effect sizes compared to first-league players (d = 0.24) at T2. Conclusion Findings emphasize great vulnerability and individuality and underline the importance of intraindividual sleep monitoring to meet the requirements needed to equally obtain health and enhance overall performance.

AbstractMaternal subclinical hypothyroidism (SCH) during pregnancy can adversely affect the neurodevelopment of the offspring. The balance of nerve growth factor (NGF)-related tropomyosin receptor kinase A/p75 neurotrophin receptor (TrkA/p75NTR) signaling in the hippocampus is important in brain development, and whether it affects cognitive function in maternal SCH’s offspring is not clear. In this study, we found that compared with the control (CON) group, expression of proliferation-related proteins [NGF, p-TrkA, phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phospho-cAMP response element-binding protein (p-CREB)] decreased in the hippocampus of the offspring in the SCH group, overt hypothyroidism (OHT) group, and the group with levothyroxine (L-T4) treatment for SCH from gestational day 17 (E17). In contrast, expression of apoptosis-related proteins [pro-NGF, p75NTR, phospho-C-Jun N-terminal kinase (p-JNK), p53, Bax and cleaved caspase-3] was increased. The two groups with treatment with L-T4 for SCH from E10 and E13, respectively, showed no significant difference compared with the CON group. L-T4 treatment enhanced relative expression of NGF by increasing NGF/proNGF ratio in offspring from maternal SCH rats. In conclusion, L-T4 treatment for SCH from early pregnancy dramatically ameliorated cognitive impairment via TrkA/p75NTR signaling, which involved activation of the neuronal proliferation and inhibition of neuronal apoptosis in SCH rats’ offspring.

Objective: This study was designed to determine the prevalence of dental caries (DMFT) in 15 years age children and to determine its association with dietary practices. Methodology: This study was conducted in the schools of Lahore having summer camp in July 2015. Study participants were 15 years old students attending summer camp by simple random technique. Written permission was taken before their examination and interview. Self-structure questionnaire was used to collect information regarding the dietary practices. DMFT index was used to measure dental caries. Data entered and analyzed using SPSS version 20. Data was assessed by percentage and frequency, descriptive part was used for demographic variables, and for dietary habits. Chi square test of association between dietary practices and dental caries was applied. P-value ≤0.05 was taken as significant. Results Total participants were sixty (n=60). Out of these 80% of the subjects of the study were males and remaining were females. Total 58.3% respondents were caries free in this study while caries positive were 41.7 %. Average DMFT score examined was 1.2. In Logistic Regression for the Prediction of Caries Status on Basis of Dietary Habits, three variables were usage of toffees, chocolates and sandwiches. These three played most important role in the production of caries status. Final regression model for calculating probability was Z= -10.665+1.479 Toffees+ 1.183 Chocolates+1.582Sandwiches. Conclusion: Findings of this study have shown that some of the dietary habits have significant relation with caries or tooth decay status like chewing gum, candies, soft drinks, sweets, toffees, chocolates and sandwiches have strong negative impact or relationship with teeth decay.

AbstractThis study investigated the acute effects of the dietary nootropic stack CILTEP®. It contains a combination of ingredients that have been individually reported to improve cognitive performance. Especially, the ingredients luteolin, which is considered a phosphodiesterase type 4 (PDE4) inhibitor, and forskolin, an adenylate cyclase stimulator, were of interest since they can increase the second messenger cAMP and thus also intracellular signaling. Numerous studies have shown that inhibition of PDE4 can improve memory in animals and humans. We examined whether acute dosing of 3 capsules of CILTEP® would improve cognitive function in healthy participants aged 30 to 40 (n = 33). We used a randomized, double-blind, placebo-controlled, two-way cross-over design. Our test battery was aimed at measuring memory performance, attention, and sensorimotor speed. The primary outcome measures were the performance on the verbal learning task and the spatial pattern separation task. Secondary outcomes included other cognitive tests, event-related potentials (ERPs), and assessment of the activity of the enzyme beta-glucuronidase and its effect on the bioavailability of luteolin, heart rate, and blood pressure. No relevant effects of acute CILTEP® treatment were found on any measure of the test battery or ERPs. Blood plasma concentrations of luteolin increased, yet about 2000 times too low to likely exert any PDE4 inhibition. CILTEP® treatment did neither affect heart rate nor blood pressure. In summary, there is no evidence that a single standardized dose of 3 capsules of CILTEP® can improve cognitive function in healthy middle-aged participants.

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. N. Szeto: None. J.A. Sosa: None. J. Vilardaga: None. J. Koh: None. W. Chang: None. Understanding the mechanisms driving parathyroid hormone (PTH) hypersecretion in primary hyperparathyroidism (PHPT) is essential for better management of this common endocrinopathy. Prior studies showed that reduced Ca2+-sensing receptor (CaSR) expression and the subsequent increases in heterodimerization of the CaSR with the type B γ-aminobutyric acid receptor 1 (GABAB1R) are causally linked to PTH hypersecretion in PHPT mouse models (Nat Metab 2:243-255). We further showed that expression of the putative GABAB1R ligands, amyloid precursor protein (APP) and its proteolytic product, β-amyloid (Aβ1-42), is significantly upregulated in adenomas of PHPT patients associated with vitamin D insufficiency/deficiency when compared to normal donor controls. The current study aims to delineate the actions of Aβ1-42 in promoting tonic PTH secretion and its interactions with vitamin D receptor (VDR) signaling in parathyroid glands (PTGs) in basal and PHPT states. We show that Aβ1-42 (200 nM) stimulated tonic PTH secretion in cultures of normal human PTGs without shifting the calcium/PTH secretion setpoint (Ca2+-setpoint). In contrast, conditional knockout (KO) of the App gene in the parathyroid cell (PTC) of PTCAppΔflox/Δflox mice significantly reduced serum PTH levels (KO: 64±13 pg/ml vs Control: 109±12 pg/ml; p&lt;0.05, n=8) despite hypocalcemia, indicating hypoparathyroidism. In PTGs cultured from the PTCAppΔflox/Δflox mice, supplementation of Aβ1-42 dose-dependently (EC50=5.6 nM, p&lt;0.001) increased PTH secretion without altering the Ca2+-setpoint. However, the stimulatory effects of Aβ1-42 on tonic PTH secretion were completely abrogated in the PTGs with concurrent deletions of App, Casr and Gabbr1 genes, supporting a direct action of Aβ1-42 on CaSR and/or GABAB1R. The latter notion is further supported by the ability of Aβ1-42 to stimulate cAMP production in cells co-expressing CaSR and GABAB1R. PTC-specific deletion of the Vdr gene in the PTCVdrΔflox/Δflox mice led to elevated serum PTH levels in vivo and increased tonic PTH secretion with unaffected Ca2+-setpoint in PTGs in vitro. Concurrent ablation of the App gene completely normalized serum PTH levels in the PTCVdrΔflox/Δflox mice and prevented PTH hypersecretion in their PTGs in culture. These findings support a critical role of the APP-derived Aβ1-42 in mediating tonic PTH secretion in the normal physiological state and suggest a new mechanism driving PTH hypersecretion in PHPT due to vitamin D deficiency. Presentation: Saturday, June 17, 2023

The Glucagon-like peptide 1 receptor (GLP-1R) is a class B1 G-protein-coupled receptor (GPCR) that is widely expressed and helps mediate insulin secretion, gastric emptying, and satiety. Currently, GLP-1R peptide agonists are used as standard of care for the treatment of T2DM and more recently for obesity. These GLP-1R peptide agonists require frequent subcutaneous injections or strict dosing guidelines thus increasing the need to develop an oral small-molecule GLP-1R agonist. Here we describe the identification of a novel small molecule GLP-1R agonist (GS-4571) which stimulated a potent cAMP response in pancreatic β-cells (EndoC-BH1) and selective agonism against human and monkey GLP-1R versus other class B GPCRs. In an intraperitoneal glucose tolerance test (IPGTT) it demonstrated improved glucose tolerance in humanized GLP-1R mice. Further studies, in obese cynomolgus monkeys, GS-4571 demonstrated similar improvements in glucose tolerance after a single dose treatment. Once-daily oral administration of GS-4571 in obese diabetic cynomolgus monkeys not only showed improved glycemic control but also showed a reduction in energy intake which led to an increase in weight loss over 36 days. Together these data support the continued development of GS-4571 into the clinic as a novel orally bioavailable GLP-1R small molecule agonist. Disclosure J. Vogel: None. M. Seung: None. B. Marchand: Employee; Gilead Sciences, Inc. Stock/Shareholder; Gilead Sciences, Inc., Pfizer Inc., Arrowhead Pharmaceuticals, Inc., Kronos Bio, Inc., Allogene Therapeutics, Inc., AbCellera Biologics Inc., Arbutus Biopharma Corporation. N. Bhangre: Employee; Gilead Sciences, Inc. Research Support; Gilead Sciences, Inc. Stock/Shareholder; Gilead Sciences, Inc. J. Mukherjee: None. S.E. Ammann: Employee; Gilead Sciences, Inc. M. Armstrong: Employee; Gilead Sciences, Inc. G. Brizgys: Employee; Gilead Sciences, Inc. E. Chin: Employee; Gilead Sciences, Inc. C. Chou: None. J. Cottell: Employee; Gilead Sciences, Inc. S.D. Schroeder: Employee; Gilead Sciences, Inc. R. Thomas-Tran: Employee; Gilead Sciences, Inc. Z. Yang: None. M. Peters: Employee; Gilead Sciences, Inc. G. Budas: Employee; Gilead Sciences, Inc. M.L. Mitchell: Employee; Gilead Sciences, Inc. M. Chojnacka: None. S. Jermain: None. J. Hung: Employee; Gilead Sciences, Inc. S. Kulkarni: None. Q. Yue: None. D.W. Lin: Employee; Gilead Sciences, Inc. D.A. Miranda: None.

Introduction & Objective: Ecnoglutide is a cAMP-biased, long-acting GLP-1RA being developed for the treatment of type 2 diabetes mellitus and obesity. Oral ecnoglutide (XW004) is formulated with an absorption enhancer, PNAC (T2026). The objective of this study was to evaluate the safety and tolerability of oral ecnoglutide in healthy adults. Methods: We conducted a randomized, double-blind, placebo-controlled Phase 1 study in healthy (Cohorts 1 to 3) and healthy obese (Cohort 4) adults. Participants (n = 56) were randomized to receive oral ecnoglutide at target doses of 7, 15, or 30 mg QD for up to 6 weeks, with dose escalation. Safety, tolerability, PK, and body weight were assessed. The results of Cohorts 1 to 4 are presented, the study is ongoing to evaluate additional dosing schemes. Results: Oral ecnoglutide was generally safe and well tolerated. The most common AEs were mild to moderate gastrointestinal events that occurred during dose escalation. There were no serious AEs. One participant experienced a Grade 3 AE of diarrhea that led to study drug discontinuation. At baseline, participants had a mean BMI of 25.8 to 26.1 kg/m2 (Cohorts 1 to 3) and 32.9 kg/m2 (Cohort 4). At end of treatment, participants in Cohorts 1 to 3 receiving up to 7, 15, or 30 mg QD oral ecnoglutide for 2 weeks had body weight change from baseline of -3.63, -3.38, and -6.55%, respectively vs -0.85% for placebo. Participants in Cohort 4 receiving up to 30 mg QD for 6 weeks had a body weight reduction of -6.76% vs -0.85% for placebo. At steady state, oral ecnoglutide 30 mg QD resulted in a plasma AUC0-24h of 12,470 h*ng/mL and calculated weekly AUC 0-168h of 87,290 h*ng/mL. Conclusion: Oral ecnoglutide was safe and well tolerated and resulted in pronounced weight loss after 6 weeks of dosing. Improved oral bioavailability enables a daily dose of 30 mg oral ecnoglutide to match or exceed the plasma exposure of weekly subcutaneous GLP-1 analogs. Oral ecnoglutide has a potential to be a best-in-class oral GLP-1RA. Disclosure Z. Zhu: None. Y. Li: Employee; Sciwind Biosciences. G. Wanjun: Employee; Sciwind Biosciences. Q. Zheng: Employee; Sciwind Biosciences. J. Deng: Employee; Sciwind Biosciences. E. Adegbite: Employee; Sciwind Biosciences. S. Ross: None. L. Telusca: None. C.L. Jones: Employee; Sciwind Biosciences. M. Fenaux: None. S. Xu: None. M.K. Junaidi: None. Funding Sciwind Biosciences

Photo by 187929822 © Victor Moussa | Dreamstime.com INTRODUCTION The nonconsensual taking of a human organ to use in transplantation medicine violates ethical principles, including autonomy, informed consent, and human rights, as well as criminal laws. When such an organ harvesting is not just nonconsensual, but performed in a way that causes a death or uses the pretense of brain death without meeting the criteria, it also violates the dead donor[1] rule.[2] The dead donor rule is both ethical and legal. It prevents organ retrieval that would predictably cause the death of the organ donor.[3] Retrieval of a vital organ is permissible only after a declaration of death.[4] Forced organ harvesting may breach the dead donor rule as it stands. A reimagined, broader dead donor rule could consider a larger timeframe in the forced organ harvesting context. In doing so, the broad dead donor rule could cover intent, premeditation, aiding and abetting, and due diligence failures. A broad definition of forced organ harvesting is ‘‘the removal of one or more organs from a person by means of coercion, abduction, deception, fraud, or abuse of power. . .’’[5] A more targeted definition is “[t]he killing of a person so that their organs may be removed without their free, voluntary and informed consent and transplanted into another person.”[6] In the global organ harvesting context, forced organ harvesting violates the World Health Organization (WHO) Guiding Principle 3, which says “live organ donors should be acting willingly, free of any undue influence or coercion.”[7] Furthermore, WHO states live donors should be “genetically, legally, or emotionally” attached to the recipient. Guiding Principle 1 applies to deceased donors, covers consent, and permits donation absent any known objections by the deceased.[8] Principle 7 says, “Physicians and other health professionals should not engage in transplantation procedures, and health insurers and other payers should not cover such procedures if the cells, tissues or organs concerned have been obtained through exploitation or coercion of, or payment to, the donor or the next of kin of a deceased donor.”[9] There are underground markets in which organ hunters prey on the local poor in countries with low wages and widespread poverty[10] and human trafficking that targets migrants for the purpose of organ harvesting.[11] This paper explores forced harvesting under the backdrop of the dead donor rule, arguing that a human rights violation so egregious requires holding even distant participants in the chain of events accountable. By interfering with resources necessary to carry out bad acts, legislation and corporate and institutional policies can act as powerful deterrents. A broader dead donor rule would highlight the premeditation and intent evidenced well before the act of organ retrieval. I. Background and Evidence In China, there is evidence that people incarcerated for religious beliefs and practices (Falun Gong) and ethnic minorities (Uyghurs) have been subjects of forced organ harvesting. A tribunal (the China Tribunal) found beyond a reasonable doubt that China engaged in forced organ harvesting.[12] Additionally, eight UN Special Rapporteurs found a system of subjecting political prisoners and prisoners of conscience to blood tests and radiological examinations to determine the fitness of their organs.[13] As early as 2006, investigators found evidence of forced organ harvesting from Falun Gong practitioners. [14] Over a million Uyghurs are in custody there, and there is ample evidence of biometric data collection.[15] An Uyghur tribunal found evidence of genocide.[16] “China is the only country in the world to have an industrial-scale organ trafficking practice that harvests organs from executed prisoners of conscience.”[17] Witnesses testified to the removal of organs from live people without ample anesthesia,[18] summonses to the execution grounds for organ removal,[19] methods of causing death for the purpose of organ procurement,[20] removing eyes from prisoners who were alive,[21] and forcing live prisoners into operating rooms.[22] The current extent of executions to harvest organs from prisoners of conscience in China is unknown. The Chinese press has suggested surgeons in China will perform 50,000 organ transplants this year.[23] Doctors Against Forced Organ Harvesting (DAFOR) concluded, “[f]orced organ harvesting from living people has occurred and continues to occur unabated in China.”[24] China continues to advertise in multiple languages to attract transplant tourists.[25] Wait times for organs seem to remain in the weeks.[26] In the United States, it is common to wait three to five years.[27] II. The Nascent System of Voluntary Organ Donation in China In China, throughout the 1990s and early 2000s, the supply of organs for transplant was low, and there was not a national system to register as a donor. A 1984 act permitted death row prisoners to donate organs.[28] In 2005, a Vice Minister acknowledged that 95 percent of all organ transplants used organs from death row prisoners.[29] In 2007 the planning of a voluntary system to harvest organs after cardiac death emerged. According to a Chinese publication, China adopted brain death criteria in 2013.[30] There had been public opposition due partly to cultural unfamiliarity with it.[31] Cultural values about death made it more difficult to adopt a universal brain death definition. Both Buddhist and Confucian beliefs contradicted brain death.[32] Circulatory death was traditionally culturally accepted.[33] The Ministry of Health announced that by 2015 organ harvesting would be purely voluntary and that prisoners would not be the source of organs.[34] There are cultural barriers to voluntary donation partly due to a Confucian belief that bodies return to ancestors intact and other cultural and religious beliefs about respect for the dead.[35] An emphasis on family and community over the individual posed another barrier to the Western approach to organ donation. Public awareness and insufficient healthcare professional knowledge about the process of organ donation are also barriers to voluntary donation.[36] Although the Chinese government claims its current system is voluntary and no longer exploits prisoners,[37] vast evidence contradicts the credibility of the voluntary transplant program in China.[38] III. Dead Donor Rule: A Source of Bioethical Debate It seems tedious to apply this ethical foundation to something as glaring as forced organ harvesting. But the dead donor rule is a widely held recognition that it is not right to kill one person to save another.[39] It acts as a prohibition on killing for the sake of organ retrieval and imposes a technical requirement which influences laws on how death is declared. The dead donor rule prevents organ harvesting that causes death by prohibiting harvesting any organ which the donor agreed to donate only after death prior to an official declaration of death. There is an ongoing ethical debate about the dead donor rule. Many in bioethics and transplant medicine would justify removing organs in specific situations prior to a declaration of death, abandoning the rule.[40] Some use utilitarian arguments to justify causing the death of someone who is unconscious and on life support irreversibly. Journal articles suggest that the discussion has moved to one of timing and organ retrieval.[41] Robert Truog and Franklin Miller are critics of the dead donor rule, arguing that, in practice, it is not strictly obeyed: removing organs while a brain-dead donor is still on mechanical ventilation and has a beating heart and removing organs right after life support is removed and cardio-pulmonary death is declared both might not truly meet the requirement of the dead donor rule, making following the rule “a dubious norm.”[42] Miller and Truog question the concept of brain death, citing evidence of whole body integrated functions that continue indefinitely. They challenge cardio-pulmonary death, asserting that the definition includes as dead, those who could be resuscitated. Their hearts could resume beating with medical intervention. Stopping life support causes death only in those whose lives are sustained by it. Some stipulate that the organ retrieval must not itself cause the death. Some would rejigger the cause of death: Daniel Callahan suggests that the underlying condition causes the death despite removal of life support.[43] But logically, a person could continue life support and be alive, so clearly, removing life support does cause death. Something else would have caused brain death or the circumstance that landed the person on mechanical ventilation. To be more accurate, one could say X caused the irreversible coma and removing life support caused the death itself. Miller and Truog take the position that because withdrawal of life support does cause death, the dead donor rule should be defunct as insincere. To them, retrieving vital organs from a technically alive donor should be permissible under limited conditions. They look to the autonomous choices of the donor or the surrogate (an autonomy-based argument). They appreciate the demand for organs and the ability to save lives, drawing attention to those in need of organs. Live donor organ retrieval arguably presents a slippery slope, especially if a potential donor is close to death, but not so close to label it imminent. They say physicians would not be obligated to follow the orders of a healthy person wishing to have vital organs removed, perhaps to save a close friend or relative. Similarly, Radcliffe-Richards, et al. argue that there is no reason to worry about the slippery slope of people choosing death so they can sell their vital organs, whether for money for their decedents or their creditors.[44] The movement toward permissibility and increased acceptance of medical aid in dying also influence the organ donation arena. The slippery slope toward the end of life has potential to become a realistic concern. Older adults or other people close to death may want to donate a vital organ, like their heart, to a young relative in need. That could greatly influence the timing of a decision to end one’s life. IV. Relating the Dead Donor Rule to Forced Organ Harvesting There is well documented evidence that in China organs have been removed before a declaration of death.[45] But one thing the dead donor rule does not explicitly cover is intent and the period prior to the events leading to death. It tends to apply to a near-death situation and is primarily studied in its relationship to organ donation. It is about death more than it is about life. Robertson and Lavee investigated data on transplantation of vital organs in China and they document cases where the declaration of death was a pretense, insincere, and incorrect. Their aim was to investigate whether the prisoners were in fact dead prior to organ harvesting.[46] (The China Tribunal found that organs have been removed from live prisoners and that organ harvesting has been the cause of death.) They are further concerned with the possible role of doctors as executioners, or at least as complicit in the execution as the organ harvesting so closely follows it. V. A Broader Dead Donor Rule A presumed ethical precursor to the dead donor rule may also be an important ethical extension of the rule: the dead donor rule must also prohibit killing a person who is not otherwise near death for the purpose of post-death organ harvesting. In China, extra-judicial killings of prisoners of conscience are premeditated ― there is ample evidence of blood tests and radiology to ensure organ compatibility and health.[47] To have effective ethical force, the dead donor rule should have an obvious application in preventing intentional killing for an organ retrieval, not just killing by way of organ retrieval. When we picture the dead donor rule, bioethicists tend to envision a person on life support who will either be taken off it and stop breathing or who will be declared brain dead. But the dead donor rule should apply to healthy people subject to persecution at the point when the perpetrator lays the ground for the later killing. At that point, many organizations and people may be complicit or unknowingly contributing to forced organ harvesting. In this iteration of the dead donor rule, complicity in its violations would be widespread. The dead donor rule could address the initial action of ordering a blood or radiology test or collecting any biometric data. Trained physicians and healthcare technicians perform such tests. Under my proposed stretch of the dead donor rule, they too would be complicit in the very early steps that eventually lead to killing a person for their organs. I argue these steps are part of forced organ harvesting and violate the dead donor rule. The donor is very much alive in the months and years preceding the killing. A conspiracy of indifference toward life, religious persecution, ethnic discrimination, a desire to expand organ transplant tourism, and intent to kill can violate this broader dead donor rule. The dead donor rule does not usually apply to the timing of the thought of organ removal, nor the beginning of the chain of events that leads to it. It is usually saved for the very detailed determination of what may count as death so that physicians may remove vital and other organs, with the consent of the donor.[48] But I argue that declaring death at the time of retrieval may not be enough. Contributing to the death, even by actions months or years in advance, matter too. Perhaps being on the deathbed awaiting a certain death must be distinguished from going about one’s business only to wind up a victim of forced organ harvesting. Both may well be declared dead before organ retrieval, but the likeness stops there. The person targeted for future organ retrieval to satisfy a growing transplant tourism business or local demand is unlike the altruistic person on his deathbed. While it may seem like the dead donor rule is merely a bioethics rule, it does inform the law. And it has ethical heft. It may be worth expanding it to the arena of human trafficking for the sake of organ removal and forced organ harvesting.[49] The dead donor rule is really meant to ensure that death was properly declared to protect life, something that must be protected from an earlier point. VI. Complicity: Meaning and Application Human rights due diligence refers to actions that people or institutions must take to ensure they are not contributing to a human rights violation. To advise on how to mitigate risk of involvement or contribution to human rights violations, Global Rights Compliance published an advisory that describes human rights due diligence as “[t]he proactive conduct of a medical institution and transplant-associated entity to identify and manage human rights risks and adverse human rights impacts along their entire value and supply chain.”[50] Many people and organizations enable forced organ harvesting. They may be unwittingly complicit or knowingly aiding and abetting criminal activity. For example, some suppliers of medical equipment and immunosuppressants may inadvertently contribute to human rights abuses in transplantation in China, or in other countries where organs were harvested without consent, under duress, or during human trafficking. According to Global Rights Compliance, “China in the first half of 2021 alone imported ‘a total value of about 24 billion U.S. dollars’ worth of medical technology equipment’, with the United States and Germany among the top import sources.”[51] The companies supplying the equipment may be able to slow or stop the harm by failing to supply necessary equipment and drugs. Internal due diligence policies would help companies analyze their suppliers and purchasers. Corporations, educational institutions, and other entities in the transplantation supply chain, medical education, insurance, or publishing must engage in human rights due diligence. The Global Rights Compliance advisory suggests that journals should not include any ill-gotten research. Laws should regulate corporations and target the supply chain also. All actors in the chain of supply, etc. are leading to the death of the nonconsenting victim. They are doing so while the victim is alive. The Stop Forced Organ Harvesting Act of 2023, pending in the United States, would hold any person or entity that “funds, sponsors, or otherwise facilitates forced organ harvesting or trafficking in persons for purposes of the removal of organs” responsible. The pending legislation states that: It shall be the policy of the United States—(1) to combat international trafficking in persons for purposes of the removal of organs;(2) to promote the establishment of voluntary organ donation systems with effective enforcement mechanisms in bilateral diplomatic meetings and in international health forums;(3) to promote the dignity and security of human life in accordance with the Universal Declaration of Human Rights, adopted on December 10, 1948; and(4) to hold accountable persons implicated, including members of the Chinese Communist Party, in forced organ harvesting and trafficking in persons for purposes of the removal of organs.[52] The Act calls on the President to provide Congress a list of such people or entities and to sanction them by property blocking, and, in the case of non-US citizens, passport and visa denial or revocation. The Act includes a reporting requirement under the Foreign Assistance Act of 1961 that includes an assessment of entities engaged in or supporting forced organ harvesting.[53] The law may have a meaningful impact on forced organ harvesting. Other countries have taken or are in the process of legal approaches as well.[54] Countries should consider legislation to prevent transplant tourism, criminalize complicity, and require human rights due diligence. An expanded dead donor rule supports legal and policy remedies to prevent enabling people to carry out forced organ harvesting. VII. Do Bioethicists Mention Human Rights Abuses and Forced Organ Harvesting Enough? As a field, bioethics literature often focuses on the need for more organs, the pain and suffering of those on organ transplant waitlists, and fairness in allocating organs or deciding who belongs on which waitlist and why. However, some bioethicists have drawn attention to forced organ harvesting in China. Notably, several articles noted the ethical breaches and called on academic journals to turn away articles on transplantation from China as they are based on the unethical practice of executing prisoners of conscience for their organs.[55] The call for such a boycott was originally published in a Lancet article in 2011.[56] There is some acknowledgement that China cares about how other countries perceive it,[57] which could lead to either improvements in human rights or cover-ups of violations. Ill-gotten research has long been in the bioethics purview with significant commentary on abuses in Tuskegee and the Holocaust.[58] Human research subjects are protected by the Declaration of Helsinki, which requires acting in the best interests of research subjects and informed consent among other protections.[59] The Declaration of Helsinki is directed at physicians and requires subjects enroll in medical research voluntarily. The Declaration does not explicitly cover other healthcare professionals, but its requirements are well accepted broadly in health care. CONCLUSION The dead donor rule in its current form really does not cover the life of a non-injured healthy person at an earlier point. If it could be reimagined, we could highlight the link between persecution for being a member of a group like Falun Gong practitioners or Uyghurs as the start of the process that leads to a nonconsensual organ retrieval whether after a proper declaration of death or not. It is obviously not ethically enough to ensure an execution is complete before the organs are harvested. It is abuse of the dead donor rule to have such a circumstance meet its ethical requirement. And obviously killing people for their beliefs or ethnicity (and extra-judicial killings generally) is not an ethically acceptable action for many reasons. The deaths are intentionally orchestrated, but people and companies who may have no knowledge of their role or the role of physicians they train or equipment they sell are enablers. An expanded dead donor rule helps highlight a longer timeframe and expanded scope of complicity. The organ perfusion equipment or pharmaceuticals manufactured in the United States today must not end up enabling forced organ harvesting. With an expanded ethical rule, the “donor is not dead” may become “the donor would not be dead if not for. . .” the host of illegal acts, arrests without cause, forced detention in labor camps, extra-judicial killings, lacking human rights due diligence, and inattention to this important topic. The expanded dead donor rule may also appeal to the bioethics community and justify more attention to laws and policies like the Stop Forced Organ Harvesting Act of 2023. - [1] The word “donor” in this paper describes any person from whom organs are retrieved regardless of compensation, force, or exploitation in keeping with the bioethics literature and the phrase “dead donor rule.” [2] Robertson, M.P., Lavee J. (2022). Execution by organ procurement: Breaching the dead donor rule in China. Am J Transplant, Vol.22,1804– 1812. doi:10.1111/ajt.16969. [3] Robertson, J. A. (1999). Delimiting the donor: the dead donor rule. Hastings Center Report, 29(6), 6-14. [4] Retrieval of non-vital organs which the donor consents to donate post-death (whether opt-in, opt-out, presumed, or explicit according to local law) also trigger the dead donor rule. [5] The Stop Forced Organ Harvesting Act of 2023, H.R. 1154, 118th Congress (2023), https://www.congress.gov/bill/118th-congress/house-bill/1154. [6] Do No Harm: Mitigating Human Rights Risks when Interacting with International Medical Institutions & Professionals in Transplantation Medicine, Global Rights Compliance, Legal Advisory Report, April 2022, https://globalrightscompliance.com/project/do-no-harm-policy-guidance-and-legal-advisory-report/. [7] WHO Guiding Principles on Human Cell, Tissue and Organ Transplantation, as endorsed by the sixty-third World Health Assembly in May 2010, in Resolution WHA63.22 https://apps.who.int/iris/bitstream/handle/10665/341814/WHO-HTP-EHT-CPR-2010.01-eng.pdf?sequence=1. [8] WHO Guiding Principles on Human Cell, Tissue and Organ Transplantation (2010). [9] WHO Guiding Principles on Human Cell, Tissue and Organ Transplantation (2010). [10] Promchertchoo, Pichayada (Oct. 19, 2019). Kidney for sale: Inside Philippines’ illegal organ trade. https://www.channelnewsasia.com/asia/kidney-for-sale-philippines-illegal-organ-trade-857551; Widodo, W. and Wiwik Utami (2021), The Causes of Indonesian People Selling Covered Kidneys from a Criminology and Economic Perspective: Analysis Based on Rational Choice Theory. European Journal of Political Science Studies, Vol 5, Issue 1. [11] Van Reisen, M., & Mawere, M. (Eds.). (2017). Human trafficking and trauma in the digital era: The ongoing tragedy of the trade in refugees from Eritrea. African Books Collective. [12] The Independent Tribunal into Forced Organ Harvesting from Prisoners of Conscience in China (China Tribunal) (2020). https://chinatribunal.com/wp-content/uploads/2020/03/ChinaTribunal_JUDGMENT_1stMarch_2020.pdf [13] UN Office of the High Commissioner, Press Release, China: UN human Rights experts alarmed by ‘organ harvesting’ allegations (UN OTHCHR, 14 June 2021), https://www.ohchr.org/en/press-releases/2021/06/china-un-human-rights-experts-alarmed-organ-harvesting-allegations. [14] David Matas and David Kilgour, Bloody Harvest. The killing of Falun Gong for their organs (Seraphim Editions 2009). [15] How China is crushing the Uyghurs, The Economist, video documentary, July 9, 2019, https://youtu.be/GRBcP5BrffI. [16] Uyghur Tribunal, Judgment (9 December 2021) (Uyghur Tribunal Judgment) para 1, https://uyghurtribunal.com/wp-content/uploads/2022/01/Uyghur-Tribunal-Judgment-9th-Dec-21.pdf. [17] Ali Iqbal and Aliya Khan, Killing prisoners for transplants: Forced organ harvesting in China, The Conversation Published: July 28, 2022. https://theconversation.com/killing-prisoners-for-transplants-forced-organ-harvesting-in-china-161999 [18] Testimony demonstrated surgeries to remove vital organs from live people, killing them, sometimes without ample anesthesia to prevent wakefulness and pain. China Tribunal (2020), p. 416-417. https://chinatribunal.com/wp-content/uploads/2020/03/ChinaTribunal_JUDGMENT_1stMarch_2020.pdf; Robertson MP, Lavee J. (2022), Execution by organ procurement: Breaching the dead donor rule in China. Am J Transplant, Vol.22,1804– 1812. doi:10.1111/ajt.16969. [19] Doctors reported being summoned to execution grounds and told to harvest organs amid uncertainty that the prisoner was in fact dead. China Tribunal (2020), p. 52-53. [20]In testimony to the China Tribunal, Dr. Huige Li noted four methods of organ harvesting from live prisoners: incomplete execution by shooting, after lethal injection prior to death, execution by removal of the heart, and after a determination of brain death prior to an intubation (pretense of brain death). China Tribunal (2020), pp. 54-55. https://chinatribunal.com/wp-content/uploads/2020/03/ChinaTribunal_JUDGMENT_1stMarch_2020.pdf [21] A former military medical student described removing organs from a live prisoner in the late 1990s. He further described his inability to remove the eyes of a live man and his witnessing another doctor forcefully remove the man’s eyes. China Tribunal (2020), p. 330. [22] In 2006, a nurse testified that her ex-husband, a surgeon, removed the eyes of 2,000 Falun Gong practitioners in one hospital between 2001 and 2003. She described the Falun Gong labor-camp prisoners as being forced into operating rooms where they were given a shot to stop their hearts. Other doctors removed other organs. DAFOH Special Report, 2022. https://epochpage.com/wp-content/uploads/sites/3/2022/12/DAFOH-Special-Report-2022.pdf [23] Robertson MP, Lavee J. (2022), Execution by organ procurement: Breaching the dead donor rule in China. Am J Transplant, Vol.22,1804– 1812. doi:10.1111/ajt.16969. [24] DAFOH Special Report, 2022. https://epochpage.com/wp-content/uploads/sites/3/2022/12/DAFOH-Special-Report-2022.pdf; DAFOH’s physicians were nominated for a Nobel Prize for their work to stop forced organ harvesting. Šućur, A., & Gajović, S. (2016). Nobel Peace Prize nomination for Doctors Against Forced Organ Harvesting (DAFOH) - a recognition of upholding ethical practices in medicine. Croatian medical journal, 57(3), 219–222. https://doi.org/10.3325/cmj.2016.57.219 [25] Robertson and Lavee (2022). [26] Stop Organ Harvesting in China, website (organization of the Falun Dafa). https://www.stoporganharvesting.org/short-waiting-times/ [27] National Kidney Foundation, The Kidney Transplant Waitlist – What You Need to Know, https://www.kidney.org/atoz/content/transplant-waitlist [28] Wu, Y., Elliott, R., Li, L., Yang, T., Bai, Y., & Ma, W. (2018). Cadaveric organ donation in China: a crossroads for ethics and sociocultural factors. Medicine, 97(10). [29] Wu, Elliott, et al., (2018). [30] Su, Y. Y., Chen, W. B., Liu, G., Fan, L. L., Zhang, Y., Ye, H., ... & Jiang, M. D. (2018). An investigation and suggestions for the improvement of brain death determination in China. Chinese Medical Journal, 131(24), 2910-2914. [31] Huang, J., Millis, J. M., Mao, Y., Millis, M. A., Sang, X., & Zhong, S. (2012). A pilot programme of organ donation after cardiac death in China. The Lancet, 379(9818), 862-865. [32] Yang, Q., & Miller, G. (2015). East–west differences in perception of brain death: Review of history, current understandings, and directions for future research. Journal of bioethical inquiry, 12, 211-225. [33] Huang, J., Millis, J. M., Mao, Y., Millis, M. A., Sang, X., & Zhong, S. (2015). Voluntary organ donation system adapted to Chinese cultural values and social reality. Liver Transplantation, 21(4), 419-422. [34] Huang, Millis, et al. (2015). [35] Wu, X., & Fang, Q. (2013). Financial compensation for deceased organ donation in China. Journal of Medical Ethics, 39(6), 378-379. [36] An, N., Shi, Y., Jiang, Y., & Zhao, L. (2016). Organ donation in China: the major progress and the continuing problem. Journal of biomedical research, 30(2), 81. [37] Shi, B. Y., Liu, Z. J., & Yu, T. (2020). Development of the organ donation and transplantation system in China. Chinese medical journal, 133(07), 760-765. [38] Robertson, M. P., Hinde, R. L., & Lavee, J. (2019). Analysis of official deceased organ donation data casts doubt on the credibility of China’s organ transplant reform. BMC Medical Ethics, 20(1), 1-20. [39] Miller, F.G. and Sade, R. M. (2014). Consequences of the Dead Donor Rule. The Annals of thoracic surgery, 97(4), 1131–1132. https://doi.org/10.1016/j.athoracsur.2014.01.003 [40] For example, Miller and Sade (2014) and Miller and Truog (2008). [41] Omelianchuk, A. How (not) to think of the ‘dead-donor’ rule. Theor Med Bioeth 39, 1–25 (2018). https://doi-org.ezproxy.cul.columbia.edu/10.1007/s11017-018-9432-5 [42] Miller, F.G. and Truog, R.D. (2008), Rethinking the Ethics of Vital Organ Donations. Hastings Center Report. 38: 38-46. [43] Miller and Truog, (2008), p. 40, citing Callahan, D., The Troubled Dream of Life, p. 77. [44] Radcliffe-Richards, J., Daar, A.S., Guttman, R.D., Hoffenberg, R., Kennedy, I., Lock, M., Sells, R.A., Tilney, N. (1998), The Case for Allowing Kidney Sales, The Lancet, Vol 351, p. 279. (Authored by members of the International Forum for Transplant Ethics.) [45] Robertson and Lavee, (2022). [46] Robertson and Lavee, (2022). [47] China Tribunal (2020). [48] Consent varies by local law and may be explicit or presumed and use an opt-in or opt-out system and may or may not require the signoff by a close family member. [49] Bain, Christina, Mari, Joseph. June 26, 2018, Organ Trafficking: The Unseen Form of Human Trafficking, ACAMS Today, https://www.acamstoday.org/organ-trafficking-the-unseen-form-of-human-trafficking/; Stammers, T. (2022), "2: Organ trafficking: a neglected aspect of modern slavery", Modern Slavery and Human Trafficking, Bristol, UK: Policy Press. https://bristoluniversitypressdigital.com/view/book/978144736. [50] Do No Harm: Mitigating Human Rights Risks when Interacting with International Medical Institutions & Professionals in Transplantation Medicine, Global Rights Compliance, Legal Advisory Report, April 2022, https://globalrightscompliance.com/project/do-no-harm-policy-guidance-and-legal-advisory-report/. [51] Global Rights Compliance, p. 22. [52] The Stop Forced Organ Harvesting Act of 2023, H.R. 1154, 118th Congress (2023). https://www.congress.gov/bill/118th-congress/house-bill/1154. [53] The Stop Forced Organ Harvesting Act of 2023, H.R. 1154, 118th Congress (2023), https://www.congress.gov/bill/118th-congress/house-bill/1154. [54] Global Rights Compliance notes that Belgium, France (passed law on human rights due diligence in the value supply chain), United Kingdom, United States, Canada, Australia, and New Zealand have legal approaches, resolutions, and pending laws. p. 45. [55] For example, Caplan, A.L. (2020), The ethics of the unmentionable Journal of Medical Ethics 2020;46:687-688. [56] Caplan, A.L. , Danovitch, G., Shapiro M., et al. (2011) Time for a boycott of Chinese science and medicine pertaining to organ transplantation. Lancet, 378(9798):1218. doi:10.1016/S0140-6736(11)61536-5 [57] Robertson and Lavee. [58] Smolin, D. M. (2011). The Tuskegee syphilis experiment, social change, and the future of bioethics. Faulkner L. Rev., 3, 229; Gallin, S., & Bedzow, I. (2020). Holocaust as an inflection point in the development of bioethics and research ethics. Handbook of research ethics and scientific integrity, 1071-1090. [59] World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects, adopted by the 18th WMA General Assembly, Helsinki, Finland, June 1964, and amended multiple times, most recently by the 64th WMA General Assembly, Fortaleza, Brazil, October 2013. https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/