A Genetic Screen in Drosophila Reveals Novel Cytoprotective Functions of the Autophagy-Lysosome Pathway

The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61α), and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1). We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms

CTRP3 and Serum Triglycerides in Children Aged 7-10 Years

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction The prevalence of obesity-related disorders has been steadily increasing over the past couple of decades. Diseases that were once only detected in adults are now prevalent in children, such as hyperlipidemia. The adipose tissue-derived hormonal factor C1q TNF Related Protein 3 (CTRP3) has been linked to triglyceride regulation especially in animal models. However, the relationship between circulating CTRP3 levels and obesity-related disorders in human subjects is controversial. CTRP3 can circulate in different oligomeric complexes: trimeric (kDa), middle molecular weight (100–300 kDa), and high molecular weight (HMW) oligomeric complexes (\u3e300 kDa). Previous work has identified that it is not the total amount of CTRP3 present in the serum, but the specific circulating oligomeric complexes that appear to be indicative of the relationship between CTRP3 and serum lipids levels. However, this work has not been examined in children. Therefore, the purpose of this study was to compare the levels of different oligomeric complexes of CTRP3 and circulating lipid levels among young children (aged 7–10 years). Methods Morphometric data and serum samples were collected and analyzed from a cross-sectional population of 62 children of self-identified Hispanic origin from a community health center, between 2015 and 2016. Serum analysis included adiponectin, insulin, leptin, ghrelin, glucagon, C-reactive peptide, triglyceride, cholesterol, IL-6, TNF, and CTRP3. Correlation analyses were conducted to explore the relationships between CTRP3 and other biomarkers. Results Total CTRP3 concentrations were significantly positively correlated with total cholesterol and HDL cholesterol. Whereas, HMW CTRP3 was not significantly associated with any variable measured. Conversely, the middle molecular weight (MMW) CTRP3 was negatively correlated with triglycerides levels, and very low-density lipoprotein (VLDL), insulin, and body mass index (BMI). The negative correlations between MMW CTRP3 and triglycerides and VLDLs were particularly strong (r2 = -0.826 and -0.827, respectively). Conclusion Overall, these data indicate that the circulating oligomeric state of CTRP3 and not just total CTRP3 level is important for understanding the association between CTRP3 and metabolic diseases. Further, this work indicates that MMW CTRP3 plays an important role in triglyceride and VLDL regulation which requires further study

The Adipokine C1q Tnf Related Protein 3 (CTRP3) Is Elevated in the Breast Milk of Obese Mothers

Background C1q TNF related protein 3 (CTRP3) is a relatively novel hormonal factor primarily derived from adipose tissue and has anti-diabetic properties. To determine if CTRP3 could play a role in early childhood development, the purpose of this study was to establish the presence of CTRP3 in breast milk (BM) and to determine whether CTRP3 levels were correlated with pregravid obesity status of the mother. Methods Breast milk was collected from breast-feeding mothers who had a pregravid body mass index (BMI) classification of normal weight (BMI 18–25 kg/m2, n = 23) or obese (BMI \u3e 30 kg/m2, n = 14). Immunoprecipitation followed by immunoblot analysis confirmed the presence of CTRP3 in BM. The concentration of CTRP3 in BM samples was determined by ELISA. Additional bioactive components were also measured by commercially available assays: ghrelin, insulin, leptin, adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and glucose. Bioactive components in normal weight and obese mothers were compared using unpaired t-test (parametric) and Mann–Whitney U-test (non-parametric), as appropriate. Results The primary findings of this study are that the adipokine CTRP3 is present in BM and CTRP3 levels are increased with pregravid obesity. Additionally, this study independently confirmed previous work that BM from obese mothers has a higher concentration of insulin and leptin. Further, no differences were observed in BM between obese and normal weight mothers in ghrelin, adiponectin, IL-6, TNF-α, or glucose levels. Conclusion This study identified a novel factor in BM, CTRP3, and showed that BM CTRP3 levels higher in obese mothers. Because of the purported insulin sensitizing effect of CTRP3, it is possible that the elevated levels of CTRP3 in the BM of obese mothers may offset negative effects of elevated leptin and insulin levels in the BM of obese mothers. Future studies will need to be conducted to determine the relevance of CTRP3 in BM and to examine the presence of other adipose tissue-derived hormonal factors

CSIRO In-Situ Lab : a multi-pronged approach to surface gas and groundwater monitoring at geological CO2 storage sites

In February 2019, at the CSIRO In-Situ Laboratory CCS project, a test was conducted where 38 t of gaseous CO2 were injected over 5 days into a fault zone at a depth of approximately 340 m. As a release test, this project enabled the testing and validation of surface and shallow well monitoring strategies at intermediate depths (i.e. depths much deeper than previous release projects and shallower than reservoirs used for CO2 storage). One of the aims of this project is to understand how CO2 would behave at intermediate depths if it did migrate from deeper depths (i.e. from a storage reservoir); the CO2 was not intended to migrate to the shallow subsurface or to surface/atmosphere. To verify that the injected CO2 remained in the subsurface, and to comply with environmental performance requirements on site, a comprehensive surface gas and groundwater monitoring program was conducted. The monitoring strategy was designed such that any leakage(s) to the surface of injected CO2 would be detected, mapped and, ultimately, quantified. The surface air monitoring program was comprised of three different but complementary approaches allowing data to be efficiently collected over different spatial and temporal scales. These approaches included continuous soil-gas chamber measurements at fixed locations, periodic soil-gas chamber measurements on gridded locations and near-surface atmospheric measurements on a mobile platform. The surface air monitoring approaches gave self-consistent results and reduced the risk of “false negative” test results. The only anomalous CO2 detected at the surface flowed from the observation well and could be directly attributed to a breach in the well casing at the injection depth providing a conduit for CO2/water to rise to the surface. Groundwater monitoring program revealed no impact on the groundwater resources attributable to the carbon injection project. Based on this work, we demonstrate that this multi-pronged monitoring strategy can be utilized to minimize the overall resources devoted to monitoring by increasing the number of monitoring approaches and diminishing the resources devoted to each technique. By maximizing the effectiveness of each element of the monitoring program, a cost-efficient and robust monitoring strategy capable of early leak detection and attribution of any leaking CO2 can be achieved

Compositional analysis of extracellular aggregates in the eyes of patients with exfoliation syndrome and exfoliation glaucoma

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-β-binding protein-2 and latent transforming growth factor-β-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-β superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease

Student Attitudes Contribute to the Effectiveness of a Genomics CURE

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student\u27s epistemic beliefs to achieving positive learning outcomes

A (not so) shallow controlled CO2 release experiment in a fault zone

The CSIRO In-Situ Laboratory Project (ISL) is located in Western Australia and has two main objectives related to monitoring leaks from a CO2 storage complex by controlled-release experiments: 1) improving the monitorability of gaseous CO2 accumulations at intermediate depth, and 2) assessing the impact of faults on CO2 migration. A first test at the In-situ Lab has evaluated the ability to monitor and detect unwanted leakage of CO2 from a storage complex in a major fault zone. The ISL consists of three instrumented wells up to 400 m deep: 1) Harvey-2 used primarily for gaseous CO2 injection, 2) ISL OB-1, a fibreglass geophysical monitoring well with behind-casing instrumentation, and 3) a shallow (27 m) groundwater well for fluid sampling. A controlled-release test injected 38 tonnes of CO2 between 336-342 m depth in February 2019, and the gas was monitored by a wide range of downhole and surface monitoring technologies. CO2 reached the ISL OB-1 monitoring well (7 m away) after approximately 1.5 days and an injection volume of 5 tonnes. Evidence of arrival was determined by distributed temperature sensing and the CO2 plume was detected also by borehole seismic after injection of as little as 7 tonnes. Observations suggest that the fault zone did not alter the CO2 migration along bedding at the scale and depth of the experiment. No vertical CO2 migration was detected beyond the perforated injection interval; no notable changes were observed in groundwater quality or soil gas chemistry during and post injection. The early detection of significantly less than 38 tonnes of CO2 injected into the shallow subsurface demonstrates rapid and sensitive monitorability of potential leaks in the overburden of a commercial-scale storage project, prior to reaching shallow groundwater, soil zones or the atmosphere. The ISL is a unique and enduring research facility at which monitoring technologies will be further developed and tested for increasing public and regulator confidence in the ability to detect potential CO2 leakage at shallow to intermediate depth

Lessons learned : the first in-situ laboratory fault injection test

The CSIRO In-Situ Laboratory has been a world first injection of CO2 into a large faulted zone at depth. A total of 38 tonnes of CO2 was injected into the F10 fault zone at approximately 330 m depth and the process monitored in detail. The site uses a well, Harvey-2, in SW Western Australia (the South West Hub CCS Project area). The top 400 m section of Harvey-2 was available for injection and instrumentation. An observation well, ISL OB-1 (400 m depth) was drilled 7 m to the north east of Harvey-2. ISL OB-1 well was cased with fibreglass to provide greater monitoring options. The CSIRO In-Situ Laboratory was designed to integrate existing facilities and infrastructure from the South West Hub CCS Project managed by the West Australian Department of Mines, Industry Regulation and Safety. While new equipment was deployed for this specific project, the site facilities were complemented by a range of mobile deployable equipment from the National Geosequestration Laboratory (NGL). The geology of the area investigated poses interesting challenges: a large fault (F10) is estimated to have up to 1000 m throw overall, the presence of packages of paleosols rather than a contiguous mudstone seal, and a 1500 m vertical thickness of Triassic sandstone as the potential commercial storage interval. This unique site provides abundant opportunities for testing more challenging geological environments for carbon storage than at other sites. While details of this first project are described elsewhere, lessons were learned during the development and execution of the project. A rigorous risk register was developed to manage project risk, but not all events encountered were foreseen. This paper describes some of the challenges encountered and the team's response. Relocation of the project site due to changes in landholder ownership) and other sensitivities resulted in the need for rapid replanning of activities at short notice resulting in the development of the site at Harvey-2. The relocation allowed other research questions to be addressed through new activities, such as the ability to consider a shallow/controlled release experiment in an extensive fault zone, but this replanning did cause some timing stress. The first test at the In-Situ Laboratory was reconfigured to address some of those knowledge gaps that shallow/controlled release experiments had yet to address. Novel approaches to drilling and completing the monitoring well also threw up unanticipated difficulties. Loss of containment from the wellbore also posed significant challenges, and the team's response to this unintended release of gas and water from the monitoring well at the conclusion of the field experiment will be discussed. Other challenges that we encountered, their impacts, and our response are also catalogued here (Table 1 and below) to enable broad knowledge exchange

A controlled CO2 release experiment in a fault zone at the in-situ laboratory in Western Australia

A controlled-release test at the In-Situ Laboratory Project in Western Australia injected 38 tonnes of gaseous CO2 between 336-342 m depth in a fault zone, and the gas was monitored by a wide range of downhole and surface monitoring technologies. Injection of CO2 at this depth fills the gap between shallow release (600 m) field trials. The main objectives of the controlled-release test were to assess the monitorability of shallow CO2 accumulations, and to investigate the impacts of a fault zone on CO2 migration. CO2 arrival was detected by distributed temperature sensing at the monitoring well (7 m away) after approximately 1.5 days and an injection volume of 5 tonnes. The CO2 plume was detected also by borehole seismic and electric resistivity imaging. The early detection of significantly less than 38 tonnes of CO2 in the shallow subsurface demonstrates rapid and sensitive monitorability of potential leaks in the overburden of a commercial-scale storage project, prior to reaching shallow groundwater, soil zones or the atmosphere. Observations suggest that the fault zone did not alter the CO2 migration along bedding at the scale and depth of the test. Contrary to model predictions, no vertical CO2 migration was detected beyond the perforated injection interval. CO2 and formation water escaped to the surface through the monitoring well at the end of the experiment due to unexpected damage to the well’s fibreglass casing. The well was successfully remediated without impact to the environment and the site is ready for future experiments

Mechanisms of TSC-mediated Control of Synapse Assembly and Axon Guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development