Background 
Cancer-induced cachexia is a complex multifactorial syndrome characterized by loss of muscle mass and function that affects up to 80% of cancer patients which contributes to the morbidity and mortality of the disease. Despite the extensive research on cancer cachexia, its exact etiology remains unknown. There is also no consensus on effective treatment for the loss of muscle mass, which subsequently impacts the tolerance and response to anti-cancer therapies. Furthermore, little is known about the effect of muscle wasting on the neuromuscular junction (NMJ), the vital synaptic connection between the motor neuron and skeletal muscle. The primary purpose of this study is to analyze the stability of the NMJs from cachectic muscles during the initiation and progression of the muscle-wasting event. To this end, we use the genetically engineered mouse model of colorectal cancer (Apc) Min/+ that has shown tumor growth and cachexia. 

Methods 
Sternomastoid, gastrocnemius, extensor digitorum longus, soleus, and diaphragm muscles were extracted from (Apc) Min/+ and age-matched controls at 16 and 24 weeks of age. The muscles were then fixed in 4% paraformaldehyde and bathed with Alexa488™ alpha-bungarotoxin to specifically label nicotinic acetylcholine receptors (nAChRs) at the postsynaptic junctional folds. Fluorescently labeled muscles were then imaged at the confocal microscope to analyze the synapse morphology, or with an inverted fluorescence microscope to quantify fluorescence intensity. The area and the density of nAChRs were then analyzed using ImageJ. To capture relevant changes in the NMJs, we performed in vivo experiments to determine the turnover rate of nAChRs at the synapse before severe weight loss occurred. To this end, we labeled sternomastoid muscles in vivo and assessed fluorescence intensity decay over time. 

Results 
At early stages of cancer-induced cachexia (16 weeks), all muscles except sternomastoid showed a significant decrease in nAChRs density in (APC) Min/+ mice compared to controls. In advanced stages of cachexia (24 weeks), all muscles from (APC) Min/+ mice showed a significant decrease in nAChRs density. All muscles showed a significant decline in the total NMJ area in (APC) Min/+ at 16 weeks and 24 weeks. 

Conclusion 
The decreased nAChR density and NMJ area in both fast and slow twitch muscles demonstrates postsynaptic NMJ alteration in cancer-induced cachectic mice. Analysis of presynaptic cells (motor neuron and Schwann cell) will be performed to further look at the pathology of the NMJ in cancer-induced cachexia, as well as younger mice to determine when the degeneration starts. 


The Impact of Cancer-induced Cachexia on the Neuromuscular Synapse

Causal pluralism proposes that humans can reason about causes and effects in terms of both dependency and process relations, depending on the scenario. Empirical support for this view is provided by responses to double prevention scenarios in which an actor attempts to bring about an outcome, a preventer attempts to prevent the outcome, and a double preventer intervenes to stop the preventer’s prevention attempt. In contrast to the predictions of the causal pluralism account, two pre-registered experiments (Ns = 400 and 450) indicate (a) that intentional actions are judged to be significantly more causative of an outcome than unintentional actions for both the actor and the double preventer and (b) that reasoners interpret the double preventer’s link to the outcome in terms of a process relation. These results underscore the need to revisit fundamental questions regarding how reasoners form and reason over representations of causal scenarios featuring intentional actions.

Revisiting Causal Pluralism: Intention, Process, and Dependency in Cases of Double Prevention

Understanding molecular mechanisms underlying physiology benefits from studies of isolated and/or cultured cells. However, virtually all experiments in cell culture are done in the absence of O2 regulation, resulting in incubator headspace O2 levels of 18-19%. In vivo, O2 levels in heart, skeletal muscle, brain, liver, and indeed most of the mammalian body, are typically 2-6%. The supra-physiological O2 levels in cell culture drive increased cellular reactive oxygen species (ROS) production, affecting many aspects of cell physiology. We have shown that supra-physiological O2 targets mitochondria, with important effects on bioenergetic function and network dynamics. Cellular responses to hormones like 17β-estradiol are significantly different in 18%, versus 5%, O2. Our RNAseq investigations show widespread effects of 18%, versus 5% O2 on diverse molecular processes ranging from DNA repair and cell cycle to energy metabolism to the viral response. Since supra-physiological O2 affects both transcriptional and translational processes, there are additional effects on relative protein abundance that are evident in proteomic analyses. The effects of supra-physiological O2 we have characterized are generally cell line-specific, making them difficult to predict. Given these myriad cellular effects of O2, it is important to reference hypoxia experiments in cell culture to physiological normoxia (e.g. 2-6%), but the vast majority of CO2 incubators used by cell culturists are not capable of regulating O2. We have developed a low-cost (&lt;1000 CAD) cell culture incubator capable of regulating O2, CO2, and temperature in their appropriate physiological ranges. Improved control over O2 levels will improve data quality in hypoxia experiments.

Importance of maintaining physioxia in cell culture

Nuclear Pore Complexes (NPCs) are large macromolecular assemblies embedded into the nuclear envelope (NE). They are involved in the selective nucleocytoplasmic transport of macromolecules, chromatin organization, and DNA repair. NPCs are organized into sub-complexes composed of repetitions of about 30 highly conserved nucleoporins (NUPs). The NUP107–160 complex (also known as Y complex) is located in the outer ring and is conserved between distantly related eukaryotes such as yeast and mammals. Here, we are going to focus on two Arabidopsis mutants deficient for this sub-complex: Atnup160 and Atnup96, also known as suppressor of auxin resistance1 (sar1) and sar3, respectively. NUP160 and NUP96 are critical for the nucleocytoplasmic transport of mRNAs and play important functions in auxin signalling, plant growth and flowering time regulation, as well as in immune defence and cold stress tolerance. In the context of our global study about the possible role of NPCs during meiosis (the specialised cell division that leads to gamete formation), we decided to explore the potential function of NUP160 and NUP96 during this cell division because the corresponding mutants show reduced fertility. The results of our study have demonstrated that meiosis is altered in a percentage of the meiocytes from these mutants, which would explain the reduction in the number of seeds. Our observations have revealed that the alteration of the Y complex causes problems in meiotic recombination, the formation of meiotic structures (such as the synaptonemal complex), and viability of the meiocytes. The possible origins of these problems will be discussed.

Exploring the function of components of the Nuclear Pore Complex during meiosis: SAR1 (NUP160) and SAR3 (NUP96)

**Abstract:**

Morphological and behavioural differences exist in many species. In rare instances these differences correspond to genetic differences at neutral molecular markers. On Kerguelen Island in the southern Indian Ocean, two species of seabirds, Kerguelen shag (Leucocarbo verrucosus) and gentoo penguin (<I>Pygoscelis papua), exhibit similar divergent behavioural and morphological patterns. Both species appear to have two main ecotypes based on foraging strategy. Colonies are either comprised of individuals who forage shallow waters or offshore in deeper waters. Differences in foraging strategy appear to be stable within a colony over time and correspond to morphological differences. On Kerguelen the differences between the ecotypes are so extreme that shags from 'inshore' colonies may be 60% lighter than individuals from 'offshore' colonies. To determine if these two foraging strategies have led to genetic differentiation, we used neutral markers to examine individuals from colonies containing the different ecotypes.

**Authors:**

Naomi Perkins¹, Juan Carlos Guevara-López², Charles-Andre Bost³, Theresa Burg¹

¹University of Lethbridge, ²Escuela Nacional de Antropología e Historia, ³Centre National de la Recherche Scientifique

Microgeographic genetic differentiation in seabirds

Early Palaeozoic represents a crucial period in Earth history, marked by intervals of life spread alternated with short-lasting crisis in biological activity. The Zagros Basin (SW Iran) could be a key area for the study of this time interval, due to the widespread and well-exposed Cambrian-Ordovician successions; nevertheless, these are still poorly constrained from a sedimentological and stratigraphic point of view. This study, carried out through a high-resolution facies and microfacies analysis of the Zard Kuh section, aims to give a contribution to the palaeogeographic evolution of this area. The studied section (296 m thick) includes the upper Mila C Member and Ilebeyk Formation, referred to the late Cambrian (Furongian) based on acritarch assemblages. The upper Mila C Member (23 m thick) is mainly characterized by medium to thin-bedded bioclastic packstones to mudstones grading upward into a nodular limestone unit. A sharp horizon marks the contact with the overlying Ilebeyk Formation, represented by a shale-dominated interval in the lower part. These mudrocks grade upward into heterolithic lithofacies, generating a coarsening upward sequence with an increase of siltstone and sandstone intercalations in the lower-middle part. The coarser-grained layers display scoured basal surfaces, with common gutter casts, capped by plane parallel lamination and hummocky cross-stratification (HCS). In the middle part of Ilebeyk Formation, siliciclastic deposits are alternated with relatively thick carbonatic intervals, showing similar features with Mila C Member. The upper part of the Ilebeyk Formation is dominated by shales, overlain at the topmost by a 28 m thick unit of micaceous sandstone and siltstone. The sandstone layers include plane-parallel bedding, trough cross-stratification and long-wavelength HCS. Based on the facies and microfacies analysis, the depositional system of the studied section is interpreted as a mixed carbonate-siliciclastic ramp. In the Mila C Member and carbonatic unit of Ilebeyk Formation, small cycles characterized by erosive surfaces and fining upward fossiliferous biomicrites suggest deposition in middle ramp setting, affected by storm events. On the other hand, the nodular limestone intervals at the top of the carbonatic units point to deposition below the SWB under a transgressional regime. The shale to heterolithic parasequences from the Ilbeyk Formation are interpreted as autocyclic fluctuations of the depositional system from lower to upper offshore environment. In the uppermost part, the sandstone-dominated interval is attributed to shoreface setting, marking an abrupt change in the bathymetric conditions after a forced regression. The sedimentological features of the studied area can be correlated with other coeval depositional events at both regional (Arabian plate) and global (Laurentia and Baltica) scales, providing new constraints for the lower Palaeozoic evolution of the northeastern Gondwanan margin.

Facies analysis and depositional environment of a late Cambrian mixed carbonate-siliciclastic ramp from the Arabian Plate (Zagros Basin, Southwestern Iran)

Epigenetic Inheritance S2

Epigenetic Inheritance S1

Comparative Biology

Synchronous neuronal activity is a hallmark of developing as well as fully developed neuronal networks and is necessary for the functional organization of the brain. However, the mechanisms of the development of these synchronous oscillations are poorly understood. While existing models assume it as a process intrinsic to neurons, recent evidence suggests that astrocytes have an important role in the development of synchronous activity. We established pure neuron only and mixed (astrocyte and neuron) cultures on multielectrode arrays (MEAs) from the embryonic chick optic tectum. Our preliminary results indicate that astrocytes are necessary for synchronous activity of neurons in culture. Mixed neuron and astrocyte cultures show random spiking activity which synchronizes over time whereas astrocyte-free neurons only show random activity without synchronization. Our model predicts that glutamate sensing at tripartite synapses via mGluRs elevates local calcium stores within astrocyte processes. With sufficient activation, the localized calcium elevation crosses a threshold causing a calcium induced calcium release (CICR) within the astrocyte leading to glutamate exocytosis. We targeted the SNARE protein Synaptobrevin (Vamp2) within astrocytes as crucial for communication with neurons via exocytotic release of glutamate. We proposed to test this model by expressing a truncated Vamp2 subunit (Vamp2 DN) which acts as a dominant negative to block exocytotic release. Astrocytes expressing the Vamp2 DN are expected to release significantly less glutamate upon calcium elevation thereby reducing synchrony of neuronal activity. We have generated astrocyte lines expressing the Synaptobrevin dominant negative (Vamp2 DN) along with the glutamate sensor iGluSnFR. We demonstrate that Vamp2 DN expressing astrocytes have significantly reduced glutamate exocytosis when CICR is induced with Ionomycin. With these tools a more comprehensive molecular model for astrocyte involvement in the generation of neuronal synchrony can be developed.

Molecular Role of Synaptobrevin & Glutamate in astrocyte-neuron interactions in the development of synchronized activity in neuronal networks

The Biomolecular Core Laboratory (BCL) is located at the Nemours/Alfred I duPont Hospital for Children in Wilmington, Delaware and has been serving the clinical and scientific community for 20 years. This resource laboratory has provided essential services in molecular biology and genetics to clinicians and research scientists at Nemours Children’s Health System and affiliated partner institutions including University of Delaware, Thomas Jefferson College of Medicine, Delaware State University, Wesley College, Delaware Technical Community College, and Christiana Care Health System. Working in close collaboration with our partnering institutions throughout the CTR and IDeA networks, our global mission is to facilitate discoveries that begin at both the basic and translational research levels. The BCL is a collaborative research lab providing support for the development of new research projects as well as mentoring, training, and educational opportunities to staff and students. We offer low-cost, fast-turnaround, high-quality services that are essential to many clinical and hypothesis driven projects. The BCL enables research excellence by offering genomics technologies including sanger sequencing, fragment analysis, nucleic acid isolation, digital PCR, genotyping, copy number analysis, gene expression analysis, as well as access to shared instrumentation. The BCL staff not only provides expertise in project design and data troubleshooting but also offers support for grant applications, budget preparations and manuscripts. The ultimate goal of this laboratory is to provide biomedical investigators with state-of-the-art technical support and clinicians with tools that can help improve pediatric supportive care and/or therapeutic intervention. This poster will present an overview of the services available to the IDeA community.

The Nemours Biomolecular Core Lab: A Resource for Biomedical Research

Humans and mice with Melanocortin 4 receptor (MC4R) deficiency remain protected from hyperglycemia despite chronic obesity and insulin resistance. We have observed that elevated glycosuria in MC4R deficient mice protects them from hyperglycemia. Moreover, our results indicate that circulating epinephrine may couple MC4R signaling with kidney glucose reabsorption. However, the direct role of epinephrine in regulating kidney glucose reabsorption remains unclear. We hypothesize that epinephrine is essential for maintaining glucose homeostasis via kidney glucose reabsorption. To test this hypothesis, we performed oral glucose tolerance tests (OGTTs) and intraperitoneal insulin tolerance tests (ITTs) in phenylethanolamine-N-methyltransferase (Pnmt) knockout (KO) mice that specifically lack epinephrine but have normal norepinephrine levels. Pnmt KO mice exhibited reduced insulin sensitivity compared to their Wild-Type (WT) littermates (Area under the curve for ITT: 9,700±256 vs. 8,482±417 mg/dL.min, p<0.05). Paradoxically, we observed improved rather than impaired glucose tolerance in Pnmt KO mice compared to their WT controls (Area under the curve for OGTT: 32,546±1,592 vs. 40,058±1,918 mg/dL.min, p<0.05). To ascertain if Pnmt KO mice, like MC4R deficient mice, show elevated glycosuria, we quantified their 24 urine glucose levels after oral glucose (250 mg) challenge. Indeed, Pnmt KO mice demonstrated elevated glycosuria compared to their WT littermates (Urine glucose: Baseline, 24.63±2.2 vs. 11.14±0.82 mg/dl; post glucose challenge: 67.83±5 vs. 16.09±1.13 mg/dl, p<0.001), again validating the phenotype similar to that of MC4R deficient mice. To determine the glucose transporters involved in mediating elevated glycosuria in the Pnmt KO mice, we measured the levels of different renal glucose transporters using western blot. We found that GLUT2 was decreased by ~26% in Pnmt KO mice compared to their WT littermates. Levels of other glucose transporters were not changed, indicating that suppression of renal GLUT2 mediates elevated glycosuria in the epinephrine deficient mice. We validated the direct effect of epinephrine on GLUT2 levels in vitro using mouse primary renal proximal tubule epithelial cells. Indeed, epinephrine selectively increased GLUT2, but did not affect other glucose transporters in the mouse kidney primary cells. Our findings establish the essential role of epinephrine in glucose reabsorption via the renal glucose transporter GLUT2. Therefore, modulating the renal adrenergic system, or, kidney-specific GLUT2 may afford alternative strategies to regulate glycosuria and ultimately mitigate diabetes.

Underline digital video library

The Impact of Cancer-induced Cachexia on the Neuromuscular Synapse

Revisiting Causal Pluralism: Intention, Process, and Dependency in Cases of Double Prevention

Importance of maintaining physioxia in cell culture

Exploring the function of components of the Nuclear Pore Complex during meiosis: SAR1 (NUP160) and SAR3 (NUP96)

Microgeographic genetic differentiation in seabirds

Facies analysis and depositional environment of a late Cambrian mixed carbonate-siliciclastic ramp from the Arabian Plate (Zagros Basin, Southwestern Iran)

Epigenetic Inheritance S2

Epigenetic Inheritance S1

Comparative Biology

Molecular Role of Synaptobrevin & Glutamate in astrocyte-neuron interactions in the development of synchronized activity in neuronal networks

The Nemours Biomolecular Core Lab: A Resource for Biomedical Research

Epinephrine Is Essential for Normal Renal Glucose Reabsorption via the Glucose Transporter GLUT2

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