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Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N-terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. Data availability. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Correspondence and requests for materials should be addressed to L.M.D.·D (lmd@unimelb.edu.au)., (Crown Copyright © 2024. Published by Elsevier Ltd. All rights reserved.)

Circadian rhythms are endogenous oscillations with approximately a 24-h period that allow organisms to anticipate the change between day and night. Disruptions that desynchronize or misalign circadian rhythms are associated with an increased risk of cardiometabolic disease. This review focuses on the liver circadian clock as relevant to the risk of developing metabolic diseases including nonalcoholic fatty liver disease (NAFLD), insulin resistance, and type 2 diabetes (T2D). Many liver functions exhibit rhythmicity. Approximately 40% of the hepatic transcriptome exhibits 24-h rhythms, along with rhythms in protein levels, posttranslational modification, and various metabolites. The liver circadian clock is critical for maintaining glucose and lipid homeostasis. Most of the attention in the metabolic field has been directed toward diet, exercise, and rather little to modifiable risks due to circadian misalignment or disruption. Therefore, the aim of this review is to systematically analyze the various approaches that study liver circadian pathways, targeting metabolic liver diseases, such as diabetes, nonalcoholic fatty liver disease, using human, rodent, and cell biology models. NEW & NOTEWORTHY Over the past decade, there has been an increased interest in understanding the intricate relationship between circadian rhythm and liver metabolism. In this review, we have systematically searched the literature to analyze the various experimental approaches utilizing human, rodent, and in vitro cellular approaches to dissect the link between liver circadian rhythms and metabolic disease.

Diastolic dysfunction is increasingly identified as a key, early onset subclinical condition characterizing cardiopathologies of rising prevalence, including diabetic heart disease and heart failure with preserved ejection fraction (HFpEF). Diastolic dysfunction characterization has important prognostic value in management of disease outcomes. Validated tools for in vivo monitoring of diastolic function in rodent models of diabetes are required for progress in pre-clinical cardiology studies. 2D speckle tracking echocardiography has emerged as a powerful tool for evaluating cardiac wall deformation throughout the cardiac cycle. The aim of this study was to examine the applicability of 2D speckle tracking echocardiography for comprehensive global and regional assessment of diastolic function in a pre-clinical murine model of cardio-metabolic disease. Type 2 diabetes (T2D) was induced in C57Bl/6 male mice using a high fat high sugar dietary intervention for 20 weeks. Significant impairment in left ventricle peak diastolic strain rate was evident in longitudinal, radial and circumferential planes in T2D mice. Peak diastolic velocity was similarly impaired in the longitudinal and radial planes. Regional analysis of longitudinal peak diastolic strain rate revealed that the anterior free left ventricular wall is particularly susceptible to T2D-induced diastolic dysfunction. These findings provide a significant advance on characterization of diastolic dysfunction in a pre-clinical mouse model of cardiopathology and offer a comprehensive suite of benchmark values for future pre-clinical cardiology studies., (© 2023. The Author(s).)

Pathological accumulation of intrahepatic triglyceride underpins the early stages of nonalcoholic fatty liver disease (NAFLD) and can progress to fibrosis, cirrhosis, and cancer of the liver. Studies in humans suggest that consumption of a diet enriched in saturated compared to unsaturated fatty acids (FAs), is more detrimental to liver fat accumulation and metabolism. However, the reasons for the divergence remain unclear and physiologically-relevant cellular models are required. Therefore, the aims of this study were to investigate the effect of modifying media composition, concentration, and treatment frequency of sugars, FAs and insulin on intrahepatocellular triglyceride content and intracellular glucose, FA and circadian function. Huh7 cells were treated with 2% human serum and a combination of sugars and FAs (low fat low sugar [LFLS], high fat low sugar [HFLS], or high fat high sugar [HFHS]) enriched in either unsaturated (OPLA) or saturated (POLA) FAs for 2, 4, or 7 days with a daily or alternating treatment regime. Stable isotope tracers were utilized to investigate basal and/or insulin-responsive changes in hepatocyte metabolism in response to different treatment regimes. Cell viability, media biochemistry, intracellular metabolism, and circadian biology were quantified. The FA composition of the media (OPLA vs. POLA) did not influence cell viability or intracellular triglyceride content in hepatocytes. In contrast, POLA-treated cells had lower FA oxidation and media acetate, and with higher FA concentrations, displayed lower intracellular glycogen content and diminished insulin stimulation of glycogenesis, compared to OPLA-treated cells. The addition of HFHS also had profound effects on circadian oscillation and gene expression. Cells treated daily with HFHS for at least 4 days resulted in a cellular model displaying characteristics of early stage NAFLD seen in humans. Repeated treatment for longer durations (≥7 days) may provide opportunities to investigate lipid and glucose metabolism in more severe stages of NAFLD., (© 2022 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Autophagy is an essential cellular process involving degradation of superfluous or defective macromolecules and organelles as a form of homeostatic recycling. Initially proposed to be a "bulk" degradation pathway, a more nuanced appreciation of selective autophagy pathways has developed in the literature in recent years. As a glycogen-selective autophagy process, "glycophagy" is emerging as a key metabolic route of transport and delivery of glycolytic fuel substrate. Study of glycophagy is at an early stage. Enhanced understanding of this major noncanonical pathway of glycogen flux will provide important opportunities for new insights into cellular energy metabolism. In addition, glycogen metabolic mishandling is centrally involved in the pathophysiology of several metabolic diseases in a wide range of tissues, including the liver, skeletal muscle, cardiac muscle, and brain. Thus, advances in this exciting new field are of broad multidisciplinary interest relevant to many cell types and metabolic states. Here, we review the current evidence of glycophagy involvement in homeostatic cellular metabolic processes and of molecular mediators participating in glycophagy flux. We integrate information from a variety of settings including cell lines, primary cell culture systems, ex vivo tissue preparations, genetic disease models, and clinical glycogen disease states., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiovascular disease is one of the leading causes of mortality in diabetes. High fructose consumption has been linked with the development of diabetes and cardiovascular disease. Serum and cardiac tissue fructose levels are elevated in diabetic patients, and cardiac production of fructose via the intracellular polyol pathway is upregulated. The question of whether direct myocardial fructose exposure and upregulated fructose metabolism have potential to induce cardiac fructose toxicity in metabolic stress settings arises. Unlike tightly-regulated glucose metabolism, fructose bypasses the rate-limiting glycolytic enzyme, phosphofructokinase, and proceeds through glycolysis in an unregulated manner. In vivo rodent studies have shown that high dietary fructose induces cardiac metabolic stress and functional disturbance. In vitro, studies have demonstrated that cardiomyocytes cultured in high fructose exhibit lipid accumulation, inflammation, hypertrophy and low viability. Intracellular fructose mediates post-translational modification of proteins, and this activity provides an important mechanistic pathway for fructose-related cardiomyocyte signaling and functional effect. Additionally, fructose has been shown to provide a fuel source for the stressed myocardium. Elucidating the mechanisms of fructose toxicity in the heart may have important implications for understanding cardiac pathology in metabolic stress settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Annandale, Daniels, Li, Neale, Chau, Ambalawanar, James, Koutsifeli, Delbridge and Mellor.)

Diabetes is associated with cardiac metabolic disturbances and increased heart failure risk. Plasma fructose levels are elevated in diabetic patients. A direct role for fructose involvement in diabetic heart pathology has not been investigated. The goals of this study were to clinically evaluate links between myocardial fructose and sorbitol (a polyol pathway fructose precursor) levels with evidence of cardiac dysfunction, and to experimentally assess the cardiomyocyte mechanisms involved in mediating the metabolic effects of elevated fructose. Fructose and sorbitol levels were increased in right atrial appendage tissues of type 2 diabetic patients (2.8- and 1.5-fold increase respectively). Elevated cardiac fructose levels were confirmed in type 2 diabetic rats. Diastolic dysfunction (increased E/e', echocardiography) was significantly correlated with cardiac sorbitol levels. Elevated myocardial mRNA expression of the fructose-specific transporter, Glut5 (43% increase), and the key fructose-metabolizing enzyme, Fructokinase-A (50% increase) was observed in type 2 diabetic rats (Zucker diabetic fatty rat). In neonatal rat ventricular myocytes, fructose increased glycolytic capacity and cytosolic lipid inclusions (28% increase in lipid droplets/cell). This study provides the first evidence that elevated myocardial fructose and sorbitol are associated with diastolic dysfunction in diabetic patients. Experimental evidence suggests that fructose promotes the formation of cardiomyocyte cytosolic lipid inclusions, and may contribute to lipotoxicity in the diabetic heart.

Type 2 diabetes is a prominent risk factor for peripheral artery disease (PAD). Yet, the mechanistic link between diabetes and PAD remains unclear. This study proposes that dysregulation of the endogenous hormone ghrelin, a potent modulator of vascular function, underpins the causal link between diabetes and PAD. Moreover, this study aimed to demonstrate the therapeutic potential of exogenous ghrelin in a diabetic mouse model of PAD. Standard ELISA analysis was used to quantify and compare circulating levels of ghrelin between (i) human diabetic patients with or without PAD (clinic) and (ii) db/db diabetic and non-diabetic mice (lab). Db/db mice underwent unilateral hindlimb ischaemia (HLI) for 14 days and treated with or without exogenous ghrelin (150 µg/kg/day.) Subsequently vascular reparation, angiogenesis, hindlimb perfusion, structure and function were assessed using laser Doppler imaging, micro-CT, microangiography, and protein and micro-RNA (miRNA) analysis. We further examined hindlimb perfusion recovery of ghrelin KO mice to determine whether an impaired vascular response to HLI is linked to ghrelin dysregulation in diabetes. Patients with PAD, with or without diabetes, had significantly lower circulating levels of endogenous ghrelin, compared to healthy individuals. Diabetic db/db mice had ghrelin levels that were only 7% of non-diabetic mice. The vascular reparative capacity of diabetic db/db mice in response to HLI was impaired compared to non-diabetic mice and, importantly, comparable to ghrelin KO mice. Daily therapeutic treatment of db/db mice with ghrelin for 14 days post HLI, stimulated angiogenesis, and improved skeletal muscle architecture and cell survival, which was associated with an increase in pro-angiogenic miRNAs-126 and -132. These findings unmask an important role for endogenous ghrelin in vascular repair following limb ischemia, which appears to be downregulated in diabetic patients. Moreover, these results implicate exogenous ghrelin as a potential novel therapy to enhance perfusion in patients with lower limb PAD, especially in diabetics.

Introduction: A person's chronotype is their entrained preference for sleep time within the 24 hours clock. It is described by the well-known concept of the 'lark' (early riser) and 'owl' (late sleeper). Evidence suggests that the 'owl' is metabolically disadvantaged due to the standard organisation of our society which favours the 'lark' and places physiological stresses on this chronotype. The aim of this study is to explore cardiometabolic health between the lark and owl in a population with an established metabolic condition - type 2 diabetes., Methods: This cross-sectional, multisite study aims to recruit 2247 participants from both secondary and primary care settings. The primary objective is to compare glycaemic control between late and early chronotypes. Secondary objectives include determining if late-chronotype is associated with poorer cardiometabolic health and other lifestyle factors, including well-being, compared with early-chronotype; describing the prevalence of the five different chronotypes in this cohort and examining the trends in glycaemic control, cardiometabolic health, well-being and lifestyle factors across chronotype., Analysis: The primary outcome (glycated haemoglobin (HbA1c)), linear regression analysis will compare HbA1c between early and late chronotypes, with and without adjustment for confounding variables. Chronotype will be modelled as a categorical variable with all five levels (from extreme-morning to extreme-late type), and as a continuous variable to calculate p for trend across the five categories. A number of models will be created; unadjusted through to adjusted with age, sex, ethnicity, body mass index, duration of diabetes, family history of diabetes, current medication and dietary habits. All secondary outcomes will be analysed using the same method., Ethics: Ethical approval from the West Midlands - Black Country Research Ethics Committee (16/WM/0457)., Dissemination: The results will be disseminated through publication in peer-reviewed medical journal, relevant medical/health conferences and a summary report sent to patients., Trial Registration Number: NCT02973412 (Pre-Results)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Significance: Energy stress in the myocardium occurs in a variety of acute and chronic pathophysiological contexts, including ischemia, nutrient deprivation, and diabetic disease settings of substrate disturbance. Although the heart is highly adaptive and flexible in relation to fuel utilization and routes of adenosine-5'-triphosphate (ATP) generation, maladaptations in energy stress situations confer functional deficit. An understanding of the mechanisms that link energy stress to impaired myocardial performance is crucial. Recent Advances: Emerging evidence suggests that, in parallel with regulated enzymatic pathways that control intracellular substrate supply, other processes of "bulk" autophagic macromolecular breakdown may be important in energy stress conditions. Recent findings indicate that cargo-specific autophagic activity may be important in different stress states. In particular, induction of glycophagy, a glycogen-specific autophagy, has been described in acute and chronic energy stress situations. The impact of elevated cardiomyocyte glucose flux relating to glycophagy dysregulation on contractile function is unknown. Critical Issues: Ischemia- and diabetes-related cardiac adverse events comprise the majority of cardiovascular disease morbidity and mortality. Current therapies involve management of systemic comorbidities. Cardiac-specific adjunct treatments targeted to manage myocardial energy stress responses are lacking. Future Directions: New knowledge is required to understand the mechanisms involved in selective recruitment of autophagic responses in the cardiomyocyte energy stress response. In particular, exploration of the links between cell substrate flux, calcium ion (Ca 2+ ) flux, and phagosomal cargo flux is required. Strategies to target specific fuel "bulk" management defects in cardiac energy stress states may be of therapeutic value.

Background: Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined., Methods: We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5-10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors., Results: CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation., Conclusions: Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.

In this study, results are reported from the analyses of vastus lateralis muscle biopsy samples obtained from a subset (n = 90) of 125 previously phenotyped, highly active male and female cyclists aged 55-79 years in regard to age. We then subsequently attempted to uncover associations between the findings in muscle and in vivo physiological functions. Muscle fibre type and composition (ATPase histochemistry), size (morphometry), capillary density (immunohistochemistry) and mitochondrial protein content (Western blot) in relation to age were determined in the biopsy specimens. Aside from an age-related change in capillary density in males (r = -.299; p = .02), no other parameter measured in the muscle samples showed an association with age. However, in males type I fibres and capillarity (p < .05) were significantly associated with training volume, maximal oxygen uptake, oxygen uptake kinetics and ventilatory threshold. In females, the only association observed was between capillarity and training volume (p < .05). In males, both type II fibre proportion and area (p < .05) were associated with peak power during sprint cycling and with maximal rate of torque development during a maximal voluntary isometric contraction. Mitochondrial protein content was not associated with any cardiorespiratory parameter in either males or females (p > .05). We conclude in this highly active cohort, selected to mitigate most of the effects of inactivity, that there is little evidence of age-related changes in the properties of VL muscle across the age range studied. By contrast, some of these muscle characteristics were correlated with in vivo physiological indices., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Objective: Diet therapy in gestational diabetes mellitus (GDM) has focused on carbohydrate restriction but is poorly substantiated. In this pilot randomized clinical trial, we challenged the conventional low-carbohydrate/higher-fat (LC/CONV) diet, hypothesizing that a higher-complex carbohydrate/lower-fat (CHOICE) diet would improve maternal insulin resistance (IR), adipose tissue (AT) lipolysis, and infant adiposity., Research Design and Methods: At 31 weeks, 12 diet-controlled overweight/obese women with GDM were randomized to an isocaloric LC/CONV (40% carbohydrate/45% fat/15% protein; n = 6) or CHOICE (60%/25%/15%; n = 6) diet. All meals were provided. AT was biopsied at 37 weeks., Results: After ∼7 weeks, fasting glucose (P = 0.03) and free fatty acids (P = 0.06) decreased on CHOICE, whereas fasting glucose increased on LC/CONV (P = 0.03). Insulin suppression of AT lipolysis was improved on CHOICE versus LC/CONV (56 vs. 31%, P = 0.005), consistent with improved IR. AT expression of multiple proinflammatory genes was lower on CHOICE (P < 0.01). Infant adiposity trended lower with CHOICE (10.1 ± 1.4 vs. 12.6 ± 2%, respectively)., Conclusions: A CHOICE diet may improve maternal IR and infant adiposity, challenging recommendations for a LC/CONV diet., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) detects Mycobacterium tuberculosis (Mtb) infection by measuring release of interferon gamma (IFN-γ) when T-cells (in heparinized whole blood) are stimulated with specific Mtb antigens. The amount of IFN-γ is determined by enzyme-linked immunosorbent assay (ELISA). Automation of the ELISA method may reduce variability. To assess the impact of ELISA automation, we compared QFT-GIT results and variability when ELISAs were performed manually and with automation., Methods: Blood was collected into two sets of QFT-GIT tubes and processed at the same time. For each set, IFN-γ was measured in automated and manual ELISAs. Variability in interpretations and IFN-γ measurements was assessed between automated (A1 vs. A2) and manual (M1 vs. M2) ELISAs. Variability in IFN-γ measurements was also assessed on separate groups stratified by the mean of the four ELISAs., Results: Subjects (N = 146) had two automated and two manual ELISAs completed. Overall, interpretations were discordant for 16 (11%) subjects. Excluding one subject with indeterminate results, 7 (4.8%) subjects had discordant automated interpretations and 10 (6.9%) subjects had discordant manual interpretations (p = 0.17). Quantitative variability was not uniform; within-subject variability was greater with higher IFN-γ measurements and with manual ELISAs. For subjects with mean TB Responses ±0.25 IU/mL of the 0.35 IU/mL cutoff, the within-subject standard deviation for two manual tests was 0.27 (CI95 = 0.22-0.37) IU/mL vs. 0.09 (CI95 = 0.07-0.12) IU/mL for two automated tests., Conclusion: QFT-GIT ELISA automation may reduce variability near the test cutoff. Methodological differences should be considered when interpreting and using IFN-γ release assays (IGRAs).

Objective: The conventional diet approach to gestational diabetes mellitus (GDM) advocates carbohydrate restriction, resulting in higher fat (HF), also a substrate for fetal fat accretion and associated with maternal insulin resistance. Consequently, there is no consensus about the ideal GDM diet. We hypothesized that, compared with a conventional, lower-carbohydrate/HF diet (40% carbohydrate/45% fat/15% protein), consumption of a higher-complex carbohydrate (HCC)/lower-fat (LF) Choosing Healthy Options in Carbohydrate Energy (CHOICE) diet (60/25/15%) would result in 24-h glucose area under the curve (AUC) profiles within therapeutic targets and lower postprandial lipids., Research Design and Methods: Using a randomized, crossover design, we provided 16 GDM women (BMI 34 ± 1 kg/m2) with two 3-day isocaloric diets at 31 ± 0.5 weeks (washout between diets) and performed continuous glucose monitoring. On day 4 of each diet, we determined postprandial (5 h) glucose, insulin, triglycerides (TGs), and free fatty acids (FFAs) following a controlled breakfast meal., Results: There were no between-diet differences for fasting or mean nocturnal glucose, but 24-h AUC was slightly higher (∼6%) on the HCC/LF CHOICE diet (P = 0.02). The continuous glucose monitoring system (CGMS) revealed modestly higher 1- and 2-h postprandial glucose on CHOICE (1 h, 115 ± 2 vs. 107 ± 3 mg/dL, P ≤ 0.01; 2 h, 106 ± 3 vs. 97 ± 3 mg/dL, P = 0.001) but well below current targets. After breakfast, 5-h glucose and insulin AUCs were slightly higher (P < 0.05), TG AUC was no different, but the FFA AUC was significantly lower (∼19%; P ≤ 0.01) on the CHOICE diet., Conclusions: This highly controlled study randomizing isocaloric diets and using a CGMS is the first to show that liberalizing complex carbohydrates and reducing fat still achieved glycemia below current treatment targets and lower postprandial FFAs. This diet strategy may have important implications for preventing macrosomia.

We present experiments on a monolayer of air-fluidized beads in which a jamming transition is approached by increasing pressure, increasing packing fraction, and decreasing kinetic energy. This is accomplished, along with a noninvasive measurement of pressure, by tilting the system and examining behavior versus depth. We construct an equation of state and analyze relaxation time versus effective temperature. By making time and effective temperature dimensionless using factors of pressure, bead size, and bead mass, we obtain a good collapse of the data but to a functional form that differs from that of thermal hard-sphere systems. The relaxation time appears to diverge only as the effective temperature to pressure ratio goes to zero.

Background: The QuantiFERON®-TB Gold In-Tube test (QFT-GIT) is a viable alternative to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, within-subject variability may limit test utility. To assess variability, we compared results from the same subjects when QFT-GIT enzyme-linked immunosorbent assays (ELISAs) were performed in different laboratories., Methods: Subjects were recruited at two sites and blood was tested in three labs. Two labs used the same type of automated ELISA workstation, 8-point calibration curves, and electronic data transfer. The third lab used a different automated ELISA workstation, 4-point calibration curves, and manual data entry. Variability was assessed by interpretation agreement and comparison of interferon-γ (IFN-γ) measurements. Data for subjects with discordant interpretations or discrepancies in TB Response >0.05 IU/mL were verified or corrected, and variability was reassessed using a reconciled dataset., Results: Ninety-seven subjects had results from three labs. Eleven (11.3%) had discordant interpretations and 72 (74.2%) had discrepancies >0.05 IU/mL using unreconciled results. After correction of manual data entry errors for 9 subjects, and exclusion of 6 subjects due to methodological errors, 7 (7.7%) subjects were discordant. Of these, 6 (85.7%) had all TB Responses within 0.25 IU/mL of the manufacturer's recommended cutoff. Non-uniform error of measurement was observed, with greater variation in higher IFN-γ measurements. Within-subject standard deviation for TB Response was as high as 0.16 IU/mL, and limits of agreement ranged from -0.46 to 0.43 IU/mL for subjects with mean TB Response within 0.25 IU/mL of the cutoff., Conclusion: Greater interlaboratory variability was associated with manual data entry and higher IFN-γ measurements. Manual data entry should be avoided. Because variability in measuring TB Response may affect interpretation, especially near the cutoff, consideration should be given to developing a range of values near the cutoff to be interpreted as "borderline," rather than negative or positive.

We report on an observation of propagating compression waves in a quasi-two-dimensional monolayer of apolar granular rods fluidized by an upflow of air. The collective wave speed is an order of magnitude faster than the speed of the particles. This gives rise to anomalously large number fluctuations, ΔN~N(0.72±0.04), which are greater than ordinary number fluctuations of N(1/2). We characterize the waves by calculating the spatiotemporal power spectrum of the density. The position of observed peaks, as a function of frequency ω and wave vector k, yields a linear dispersion relationship in the long-time, long-wavelength limit and a wave speed c=ω/k. Repeating this analysis for systems at different densities and air speeds, we observe a linear increase in the wave speed with increasing packing fraction with almost no dependence on the air flow. We also observe that the parallel and perpendicular root-mean-square speeds of the rods are identical when waves are present, but become different at low packing fractions where there are no waves. Based on this apparent exclusivity, we map out the phase behavior for the existence of waves vs speed anisotropy as a function of density and fluidizing air flow.

We study a quasi-two-dimensional monolayer of granular rods fluidized by a spatially and temporally homogeneous upflow of air. By tracking the position and orientation of the particles, we characterize the dynamics of the system with sufficient resolution to observe ballistic motion at the shortest time scales. Particle anisotropy gives rise to dynamical anisotropy and superdiffusive dynamics parallel to the rod's long axis, causing the parallel and perpendicular mean-square displacements to become diffusive on different time scales. The distributions of free times and free paths between collisions deviate from exponential behavior, underscoring the nonthermal character of the particle motion. The dynamics show evidence of rotational-translational coupling similar to that of an anisotropic Brownian particle. We model rotational-translational coupling in the single-particle dynamics with a modified Langevin model using nonthermal noise sources. This suggests a phenomenological approach to thinking about collections of self-propelling particles in terms of enhanced memory effects.

We report a case of a 44-year old female who presented to her physician complaining of mild dyspnea. A follow-up chest X-ray and chest computed tomography scan revealed a giant bilateral intrathoracic mass, filling the right thoracic cavity and extending across the anterior mediastinum into the left chest cavity. This large mass caused a marked shift in the midline structures, displacing the heart to the left hemi-thorax. The patient underwent surgical removal of the thoracic and breast mass, with histologic examination confirming the diagnosis of a giant intrathoracic lipoma, weighing 4,320 grams and measured 34 x 28 x 11 cm. It is the largest intrathoracic lipoma documented in the modern literature.

Background: Interferon-gamma release assays (IGRAs) are attractive alternatives to the tuberculin skin test (TST) for detecting Mycobacterium tuberculosis infection. However, the inability to definitively confirm the presence of most M. tuberculosis infections hampers assessment of IGRA accuracy. Although IGRAs are primarily indicated for the detection of latent tuberculosis infection, we sought to determine the sensitivity of the TST and 2 whole-blood IGRAs (QuantiFERON-TB assay [QFT] and QuantiFERON-TB Gold assay [QFT-G]) in situations in which infection is confirmed by recovery of M. tuberculosis by culture., Methods: We conducted a prospective, multicenter, cross-sectional comparison study in which 148 persons suspected to have tuberculosis were tested simultaneously with the TST, QFT, and QFT-G., Results: M. tuberculosis was cultured from samples from 69 (47%) of 148 persons suspected to have tuberculosis; the TST induration was > or = 5 mm for 51 (73.9%) of the 69 subjects (95% confidence interval [CI], 62.5%-82.8%). The QFT indicated tuberculosis infection for 48 (69.6%) of the 69 subjects (95% CI, 57.9%-79.2%) and was indeterminate for 7 (10.1%). The QFT-G yielded positive results for 46 (66.7%) of the 69 subjects (95% CI, 54.9%-76.7%) and indeterminate results for 9 subjects (13.0%). If subjects with indeterminate QFT-G results were excluded, 46 (76.7%) of 60 subjects (95% CI, 64.6%-85.6%) had positive TST results, and the same number of subjects had positive QFT-G results. HIV infection was associated with false-negative TST results but not with false-negative QFT-G results., Conclusions: The TST, QFT, and QFT-G have similar sensitivity in persons with culture-confirmed infection. As with the TST, negative QFT and QFT-G results should not be used to exclude the diagnosis of tuberculosis in persons with suggestive signs or symptoms.

Background: Military personnel are at risk for acquiring Mycobacterium tuberculosis infection because of activities in close quarters and in regions with a high prevalence of tuberculosis (TB). Accurate tests are needed to avoid unnecessary treatment because of false-positive results and to avoid TB because of false-negative results and failure to diagnose and treat M. tuberculosis infection. We sought to estimate the specificity of the tuberculin skin test (TST) and 2 whole-blood interferon-gamma release assays (QuantiFERON-TB assay [QFT] and QuantiFERON-TB Gold assay [QFT-G]) and to identify factors associated with test discordance., Methods: A cross-sectional comparison study was performed in which 856 US Navy recruits were tested for M. tuberculosis infection using the TST, QFT, and QFT-G., Results: Among the study subjects, 5.1% of TSTs resulted in an induration > or = 10 mm, and 2.9% of TSTs resulted in an induration > or = 15 mm. Eleven percent of QFT results and 0.6% of QFT-G results were positive. Assuming recruits at low risk for M. tuberculosis exposure were not infected, estimates of TST specificity were 99.1% (95% confidence interval [CI], 98.3%-99.9%) when a 15-mm cutoff value was used and 98.4% (95% CI, 97.3%-99.4%) when a 10-mm cutoff value was used. The estimated QFT specificity was 92.3% (95% CI, 90.0%-94.5%), and the estimated QFT-G specificity was 99.8% (95% CI, 99.5%-100%). Recruits who were born in countries with a high prevalence of TB were 26-40 times more likely to have discordant results involving a positive TST result and a negative QFT-G result than were recruits born in countries with a low prevalence of TB. Nineteen (50%) of 38 recruits with this type of discordant results had a TST induration > or = 15 mm., Conclusions: The QFT-G and TST are more specific than the QFT. No statistically significant difference in specificity between the QFT-G and TST was found using a 15-mm induration cutoff value. The discordant results observed among recruits with increased risk of M. tuberculosis infection may have been because of lower TST specificity or lower QFT-G sensitivity. Negative QFT-G results for recruits born in countries where TB is highly prevalent and whose TST induration was > or = 15 mm suggest that the QFT-G may be less sensitive than the TST. Additional studies are needed to determine the risk of TB when TST and QFT-G results are discordant.

Background: Thoracoscopic lobectomy is emerging as a potential alternative to thoracotomy for early stage lung cancer. The issues of safety and oncologic efficacy should be analyzed before recommending this procedure for widespread use., Methods: Thoracoscopic lobectomy was attempted in 110 consecutive patients (age, 35 to 81 years) with tumors that were judged to be amenable to lobectomy over a 26-month period. Exclusion criteria included tumors greater than 5 cm in diameter, T3 tumors, endobronchial tumors visible at bronchoscopy, the use of induction therapy, extensive N1 disease on computed tomographic scan, and N2 disease at mediastinoscopy. The procedures were performed without rib spreading using two ports and included anatomic hilar dissection and individual vessel stapling., Results: Thoracoscopic lobectomy and mediastinal lymph dissection was successfully performed in 108 patients (98.2%); 2 patients required conversion to thoracotomy to control bleeding in the setting of dense hilar adenopathy. There were no intraoperative deaths and 4 perioperative deaths (3.6%) caused by pneumonia and associated adult respiratory distress syndrome (3 patients) and stroke (1 patient). Major complications included pneumonia (5 patients), stroke (1 patient), and return to the operating room to revise the bronchial closure (1 patient). Minor complications included prolonged air leak (6 patients), atrial fibrillation (4 patients), blood transfusion (2 patients) and ileus (1 patient). Median time to chest tube removal was 3 days, and median length of stay was 3 days., Conclusions: Thoracoscopic lobectomy is a safe and effective strategy for patients with early stage lung cancer. Long-term follow-up is required to determine if recurrence rate and 5-year survival are comparable with thoracotomy for lobectomy.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Organs transplanted between phylogenetically-disparate species, such as from the pig into the primate, are subject to hyperacute and acute vascular rejection. Hyperacute rejection of a porcine organ by a primate is thought to be initiated by the binding of xenoreactive natural antibodies to Galalpha1-3Gal expressed on the endothelial lining of blood vessels in the xenograft. The factor(s) which initiates acute vascular rejection is uncertain; however, there is some evidence implicating xenoreactive antibodies. The nature of the humoral response which might contribute to acute vascular rejection of a porcine organ was investigated in baboons which received a porcine cardiac xenograft plus immunosuppression with methylprednisolone, azathioprine, and cyclosporine. Following rejection and surgical removal of the xenografts, the serum concentration of xenoreactive antibodies increased in untreated animals but in immunosuppressed animals was similar to the concentration in preimmune serum. The antibodies in the sensitized recipients were specific for Galalpha1-3Gal (70-95%) and other determinants (5-30%). However, cross-blocking studies showed that, following xenotransplantation, the immunosuppressed baboons had no detectable IgM or IgG directed against "new" endothelial antigens. These results indicate that antibodies made by immunosuppressed individuals in response to xenotransplantation are much like xenoreactive natural antibodies and suggest that acute vascular rejection might in some cases be addressed by therapeutic strategies aimed at those antibodies.

Xenoreactive natural antibodies in humans and higher primates are directed predominantly at Gal alpha 1-3Gal. These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts. The contribution of anti-Gal alpha 1-3Gal antibodies to the xenoractive natural antibody repertoire and to the initiation of hyperacute rejection was tested in a pig-to-baboon cardiac xenograft model. Anti-Gal alpha 1-3Gal antibodies were depleted from baboons by extracorporeal absorption of anti-Gal alpha 1-3Gal antibodies from plasma using columns with a matrix bearing Gal alpha 1-3Galb1-4GlcNAc. Specific removal of anti-Gal alpha 1-3Gal antibodies was achieved prior to transplantation as demonstrated by immunoassay. Porcine hearts were then transplanted into these baboons and the outcome of the transplants was analysed. Immunofluorescence revealed little deposition of baboon antibodies in the grafts. The porcine hearts did not undergo hyperacute rejection even though complement activity was approximately 90% of baseline at the time of transplantation. These findings demonstrate that anti-Gal alpha 1-3Gal antibodies constitute a major fraction of xenoreactive natural antibodies in primate blood and that these antibodies contribute significantly to the pathogenesis of hyperacute xenograft rejection.

An animal model which allows for the implantation and development of intracutaneous melanoma is described. Intradermal vesicles are created by a negative pressure apparatus in DBA/2J mice. S91 melanoma cells are injected into the blister cavities. Tumor growth occurs within 7 days and the yield approaches 100%. This model is a rapid, efficient, and applicable system for the study of melanoma growth dynamics and of the effects of systemic and topical anti-tumor agents on melanoma proliferation.