Breeding Biology Of Chuck-Will\u27s-Widows: Incubation, Brooding, And Provisioning Behavior And Characteristics Of Nest Sites

Chuck-will\u27s-widows (Antrostomus carolinensis) are cryptically colored grounding-nesting nightars that breed throughout much of the eastern United States, primarily in mixed-forest habitat. Because of their cryptic plumage and nocturnal habits, little is known about their behavior, particularly their breeding and nesting behavior. Thus, my objectives were to: (1) quantify patterns of incubation behavior (e.g., on bouts vs. off bouts) and the respective roles of males and females, (2) quantify the brooding and provisioning behavior of males and females, and, (3) compare the characteristics of nest sites and randomly selected unused sites to determine those features important in nest site selection. My study was conducted at the Richard and Lucile Durrell Edge of Appalachia Preserve in Adams County, Ohio. During the breeding seasons of 2011 and 2012, I located six Chuck-will\u27s-widow nests. All clutches consisted of two eggs laid on either leaf litter (N = 4) or bare ground (N = 2). Females were observed incubating more often (N = 12 observations at four nests) than males (N = 3 observations at two nests). Additionally, during 16 visits to five nests, females were flushed from nests more often (N = 14, or 87.5% of visits) than males (N = 2). Overall, the mean duration of incubation on-bouts was 442.1 min (N = 94). Incubation bouts of females were longer (mean = 274.5 min, N = 12 at four nests) than those of males (mean = 7.7 min, N = 3 at two nests). Females incubated eggs both during the day and at night whereas males were only observed incubating eggs at dusk. Most incubation off-bouts occurred at dawn (N = 45 at six nests) and dusk (N = 48 at six nests) and averaged 35.0 min in duration (N = 104). Nightjars are visually oriented crepuscular and nocturnal insectivores so eggs are likely left unattended at dusk and dawn so adults can forage. Off-bouts were shorter in duration during the middle (days 10 - 15) and late (days 16-21) stages of incubation than early in incubation (days 4 - 9). Changes in nest attentiveness may be related to the increased reproductive value of eggs as the incubation period progresses. Both males and females provisioned young, with eight observed visits by males and three by females. At dusk, feeding rates were highest (P = 0.0026) during the hour immediately after sunset and declined thereafter. Chuck-will\u27s-widows may actively forage during the period after sunset because prey availability is higher and light levels are sufficient for effective foraging. Most characteristics of nest sites and randomly selected unused sites were similar, but nest sites had less canopy cover (mean = 70.8%) than unused sites (mean = 92.4%). Nest sites in areas with less canopy cover may have increased light levels and thus may provide better foraging habitat for Chuck-will\u27s-widows. In addition, a more open canopy may make it easier for adults flying to and from nest sites. Chuck-will\u27s-widows have high aspect ratio wings and so may prefer more open canopies above nest sites to make it easier to visit and leave nests under low light conditions

Obligatory Leaf Litter Spiders: An Ignored Guild?

There are over 100,000 described species of arachnids in the world; these include spiders, scorpions, ticks, mites, etc. There are some 45,000 described species of spiders worldwide, with 3,800 species known from within the United States. Spiders can be classified into several guilds, including sensing web weavers, sheet web weavers, space web weavers, orb web weavers, specialists, ambush hunters, and ground hunters. A guild for spiders is defined as a group of spiders that exploit similar resources using similar behaviors in the same habitat. We are interested in assessing the spiders that are obligatory leaf litter dwellers – these are spiders that spend their entire lives within the tight spaces of the forest floor leaf litter. These spiders, as adults are extremely small, 1-3 mm in length, and only occur in a habitat that is largely ignored by most arachnologists. Therefore, for this project, we are interested in assessing the biodiversity of spiders that are obligatory leaf litter inhabitants. Obligatory leaf litter spiders may represent an ignored guild of spiders. To date, we have identified 13 families of leaf litter dwelling spiders, but only five families we consider obligatory leaf litter inhabitants. These include spiders from the families Cybaeidae, Gnaphosidae, Liocranidae, Lycosidae, and Salticidae. We are only examining mature male and female spiders since these are most likely obligatory leaf litter dwellers. The most abundant families include both Liocranidae and Lycosidae, with Salticidae and Gnaphosidae being the least abundant.https://scholarworks.moreheadstate.edu/celebration_posters_2023/1043/thumbnail.jp

Immunity and tolerance to the tumor-associated antigen MUC1

Human mucin 1 (MUC1) is a highly glycosylated transmembrane glycoprotein that is expressed on the luminal surface of ductal epithelial cells. Human adenocarcinomas overexpress MUC1 as a tumor-associated antigen (TAA) that presents to the immune system peptide epitopes and glycopeptide epitopes with tumor specific carbohydrates, such as mono- and disaccharides known as Tn, sialyl-Tn, and TF antigens. Studies in MUC1 transgenic (MUC1-Tg) mice have indicated that, compared to transgene negative wild-type (WT) mice, the MUC1-Tg immune system maintains a certain level of tolerance to the MUC1 peptide, reflected most notably in decreased CD4 T cell help. We made a novel observation that in contrast to suppressed responses to the MUC1-peptide vaccine, vaccination of MUC1-Tg mice with tumor-associated MUC1 glycopeptide resulted in effective anti-MUC1 immunity similar to that elicited in WT mice. We hypothesized that the tumor-associated glycopeptides were seen as foreign and therefore not subject to tolerance. To study CD4 T cell responses to MUC1 glycopeptides we generated glycopeptide GST(GalNAc;Tn)A specific, MHC-Class II restricted CD4 T cell hybridoma, RF6. We cloned the RF6 TCR (RFT; Vá4.1Já16-Vâ15Jâ1.3) and generated TCR transgenic mice RFT-Tg. Using the RFT-Tg mice and the previously generated MUC1 peptide-specific TCR transgenic VFT-Tg mice, we show that peptide-specific VFT CD4 T cells transferred to MUC1-Tg mice are suppressed through mechanisms of peripheral tolerance, which are not induced against MUC1-glycopeptide specific RFT CD4 T cells. We show that peptide-specific CD4 T cells are transiently activated upon transfer into MUC1-Tg mice, suggesting that MUC1-peptide epitope is presented in the periphery in healthy mice as well as in tumor bearing mice. In contrast, MUC1-glycopeptide epitopes are tumor specific and thus treated as foreign antigens in MUC1-Tg mice, resulting in effective activation of glycopeptide-specific CD4 T cells. Simultaneous activation of glycopeptide-specific T cells can break tolerance of peptide-specific T cells. Our findings with MUC1 apply to other TAA that contain some epitopes that are "self" and subject to self tolerance and other epitopes that are tumor specific ("foreign") and not affected by tolerance. Understanding this distinction is very important in the development of effective and safe cancer vaccines

Autophagy is Required for mTOR-Mediated Anabolism in Skeletal Muscle

PURPOSE: While much has been discovered about the role autophagy in protein degradation, recent evidence suggests that autophagy is required for muscular adaptations to exercise, hinting at a hitherto unknown cross-talk between autophagic proteolysis and muscle protein anabolism. Here, we set out to further elucidate the metabolic mechanisms by which autophagy may influence protein anabolism. METHODS: L6 myoblasts received either electrical pulse stimulation (EPS) to induce muscle contraction or were unstimulated to serve as controls, and were then treated with an inhibitor of the ATG4 enzyme which catalyzes the initial step of autophagy NSC185058 (NSC, 100 μM) or DMSO as a vehicle control (VC). After 24 hours, cells were lysed and Western immunoblotted for P70S6K, DEPTOR, MAPK, AMPK, LC3, and P62. Differences between VC and NSC treated groups were assessed by a two-tailed t-test, while comparisons between VC, EPS, and EPS+NSC groups were made using one-way ANOVA and SNK post-hoc test, with α levels set at 0.05. RESULTS: EPS induced a 97% increase in P70S6K phosphorylation (p\u3c0.05), with NSC treatment blunting this effect, leading to a 22% increase (P\u3e0.05). EPS resulted in a 37% reduction in DEPTOR content (p\u3c0.05); however, NSC treatment alone produced a 166% decrease in DEPTOR level (p\u3c0.05), with EPS+NSC leading to an even larger reduction (-766%) in DEPTOR than EPS alone. NSC treatment led to a decrease (-85%, p\u3e0.05) LC3II/I ratio relative to VC, which was reduced in both the EPS (-68%, p\u3c0.05) and EPS+NSC (-87%, p\u3c0.05) conditions. P62 content increased by 749% with EPS (p\u3c0.05), with no significant difference in P62 level between VC and EPS+NSC, and NSC treatment alone led to a 61% decrease in P62 (p\u3c0.05). MAPK phosphorylation was elevated in both EPS (99.9%, p\u3e0.05) and EPS+NSC (149.13, p\u3c0.05). Neither NSC nor EPS+NSC altered phosphorylation status of AMPK. CONCLUSION: Despite reductions in DEPTOR, mTOR activity was blunted in EPS+NSC cells, indicating that mTOR mediated anabolic signaling requires autophagy post muscle contraction. This is particular to the mTOR pathway, as an increase in MAPK phosphorylation was still observed in EPS+NSC. While the decrease in LC3II/I ratio and accumulation of P62 seen after EPS are likely due to inhibition of autophagy due to mTOR activity, our data indicate that inhibition of ATG4 by NSC185058 blunts mTOR activity after muscle contraction. This effect is not due to activation of the cellular energy sensor AMPK, as we found no increase in AMPK phosphorylation in any condition. Further work will be required to fully elucidate the mechanism by which NSC185058 inhibits mTOR-mediated anabolism

Autophagy, but Not Proteolysis, May Aid in Muscle Protein Synthesis

For muscle growth to occur, protein synthesis must be greater than protein degradation. However, up to this point, anabolic pathways have garnered the brunt of investigations examining anabolic capacity with little investigation into the connectedness of catabolic signaling on these anabolic targets. PURPOSE: The purpose of this study was to elucidate the contributions of proteasomal-dependent and autophagic-dependent catabolic pathways on anabolism via analysis of fractional synthetic rates (FSR) in L6 myotubes. METHODS: Differentiated, cultured L6 myoblasts were treated with media containing 4% deuterium oxide (stable isotope label) and a corresponding pharmacological treatment (NSC 185058 [autophagic inhibitor; 100 μM], MG-262 [proteasomal inhibitor; 0.01 μM] or DMSO control; n=3/group) during the final 24-hours of the differentiation period prior to harvest. The myofibrillar pellet of the processed samples was used to determine FSR via mass-spectrometry analysis. DMSO-treated myotubes served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences among groups. RESULTS: Our results indicate that MG-262 had no impact on myofibrillar FSR when compared to DMSO control (MG-262 1.0993 %/day vs. control 1.239 %/day). However, NSC 185058 lowered myofibrillar FSR (NSC 185058 0.9009 %/day vs. control 1.239 %/day; P=0.0282). CONCLUSION: These data suggest that inhibition of autophagic machinery can impair anabolism. This may be due to autophagy’s role in increasing the amino acid pool within the cell. Further, the lack of inhibition seen from MG-262 suggests that there is a delineation of roles within the catabolic pathways in regard to their influence on anabolism in healthy, metabolically unchallenged myotubes

Insulin-induced Increase in Anabolic Capacity is Blunted by Autophagic Inhibition in L6 Myotubes

Insulin is an anabolic hormone that acts on skeletal muscle cells to stimulate protein synthesis, an effect that is enhanced by the availability of amino acids. While autophagy within the cell provides an intracellular source of amino acids to support anabolism, little is known about how this pathway impacts the insulin-induced increase in anabolic capacity within skeletal muscle cells. PURPOSE: The purpose of this study was to determine the impact of autophagic inhibition in cultured L6 myotubes in conjunction with insulin stimulation in vitro. METHODS: Differentiated, cultured L6 myotubes were treated for 24 hours with or without insulin (100 nM) and NSC 185058 (100 μM), a specialized inhibitor of the autophagic catabolic pathway, in media enriched with 4% deuterium. Cells were harvested from each treatment group (n=3/group) 24 hours post-deuterium enrichment and were processed for protein synthesis and western blot protein analysis. A one-way ANOVA was used to compare groups, and when significant F ratios were present, a Student’s Newman-Keuls post hoc procedure was used to test differences among group means. Alpha was set at p≤0.05 for all analyses. RESULTS: Cells treated with insulin (INS) had a higher ratio of phosphorylated to total P70S6K compared to untreated (CON) cells and those incubated with both insulin and NSC 185058 (INS+NSC; 1694% and 327%, respectively; p\u3c0.05). INS+NSC also decreased the ratio of phosphorylated to total 4EBP1 relative to CON (-51%) and INS (-49%), although these differences were not significant (p\u3e0.05). Myofibrillar protein synthesis was stimulated with INS compared to CON and INS+NSC (30.3% and 70.1% respectively; p\u3c0.05) but was lower in INS+NSC relative to CON (-23.4%; p\u3c0.05). CONCLUSION: Results from our study indicate that insulin (100 nM) stimulates anabolism in skeletal muscle cells, but that addition of the autophagic inhibitor NSC 185058 (100 μM) blunts this effect to a level similar to or less than control. Further, our data suggest that the reduction of protein synthesis is mediated through the downregulation of the mTORC1 signaling pathway. While it is widely recognized that insulin promotes anabolic activity through both the direct stimulation of mTOR signaling and extracellular amino acid uptake, our data strongly indicate that autophagic processes are necessary for full anabolic responses in muscle. This decrease in anabolic capacity supports previous literature indicating that the amino acid availability impacts the stimulatory impact of insulin on protein synthesis

Early postnatal EEG features of perinatal arterial ischaemic stroke with seizures

Background: Stroke is the second most common cause of seizures in term neonates and is associated with abnormal long-term neurodevelopmental outcome in some cases. Objective: To aid diagnosis earlier in the postnatal period, our aim was to describe the characteristic EEG patterns in term neonates with perinatal arterial ischaemic stroke (PAIS) seizures. Design: Retrospective observational study. Patients: Neonates >37 weeks born between 2003 and 2011 in two hospitals. Method: Continuous multichannel video-EEG was used to analyze the background patterns and characteristics of seizures. Each EEG was assessed for continuity, symmetry, characteristic features and sleep cycling; morphology of electrographic seizures was also examined. Each seizure was categorized as electrographic-only or electroclinical; the percentage of seizure events for each seizure type was also summarized. Results: Nine neonates with PAIS seizures and EEG monitoring were identified. While EEG continuity was present in all cases, the background pattern showed suppression over the infarcted side; this was quite marked (>50% amplitude reduction) when the lesion was large. Characteristic unilateral bursts of theta activity with sharp or spike waves intermixed were seen in all cases. Sleep cycling was generally present but was more disturbed over the infarcted side. Seizures demonstrated a characteristic pattern; focal sharp waves/spike-polyspikes were seen at frequency of 1-2 Hz and phase reversal over the central region was common. Electrographic-only seizure events were more frequent compared to electroclinical seizure events (78 vs 22%). Conclusions: Focal electrographic and electroclinical seizures with ipsilateral suppression of the background activity and focal sharp waves are strong indicators of PAIS. Approximately 80% of seizure events were the result of clinically unsuspected seizures in neonates with PAIS. Prolonged and continuous multichannel video-EEG monitoring is advocated for adequate seizure surveillance

Autophagy is Required for the Anabolic Response to Muscle Contraction

Exercise is a key stimulus in regulating the behavior and metabolism of skeletal muscle, with exercise inducing muscular growth through activation of the anabolic mechanistic target of rapamycin kinase (mTOR). Separately, there is mounting evidence that exercise increases autophagy (one of the main routes by which intracellular proteins are degraded) and that the autophagic process may indeed be required for adaptations to exercise training. PURPOSE: To investigate the effects of autophagy inhibition on mTOR signaling and cellular anabolism after muscular contraction. METHODS: Cultured L6 myotubes were to exposed to electrical pulse stimulation using a stimulator set to deliver bipolar pulses of 30V at 100 Hz for 200 ms every fifth second for 60 minutes. Subsequently, cells received either vehicle control, or 100 μM NSC-185058, an antagonist of the key autophagy protein ATG4B and known inhibitor of autophagy. All groups were also exposed to 4% deuterium oxide, a stable isotopic tracer for measurements of protein synthesis. 24 hours post “exercise” bout, cells were lysed in ice-cold Norris buffer, and prepared for Western immunoblot of protein expression, or determination of protein fractional synthesis rate (FSR) of the myofibrillar fraction via mass-spectrometry analysis. Non-stimulated cells receiving vehicle control treatment served as controls, with a one-way analysis of variance and Tukey’s post-hoc test used to test for any differences between groups. RESULTS: We found that phosphorylation of a key downstream target of mTOR, P70S6 kinase, was roughly seven times greater in cells subjected to EPS and vehicle control (710.3%) relative to control (p0.05). While there was a trend for EPS treatment to increase expression of ATG4B, along with a reduction of ATG4B content as a result of NSC-185058 treatment, this finding did not rise to the level of statistical significance. There were no differences in FSR between cells exposed to EPS; however, NSC-185058 treatment significantly reduced FSR in EPS treated cells relative to controls (0.8712 %/hr vs 1.193 %/hr). CONCLUSION: These findings present two conclusions: high-intensity EPS as an in vitro model of exercise elevates mTOR signaling through P70S6K 24 hours post exercise, and mTOR activation as a result of muscular contraction is reliant upon autophagy in skeletal muscle. Further work will be required to elucidate the dynamics of this relationship, and the interplay between skeletal muscle autophagy and anabolism