Sympathetic innervation regulates basement membrane thickening and pericyte number in rat retina

PURPOSE. To determine whether loss of sympathetic innervation alters basement membrane thickness and pericyte loss. METHODS. Sympathetic innervation to the eye was destroyed by surgical removal of the right superior cervical ganglion in rats. Basement membrane changes were assessed by real-time PCR and electron microscopy. The number of pericytes was measured by immunofluorescent staining for NG2 proteoglycan. Steady-state mRNA levels were also evaluated for platelet-derived growth factor-BB (PDGF-BB). RESULTS. Loss of sympathetic innervation caused a significant increase in steady state mRNA levels of fibronectin and a 15% increase in laminin-␤1 mRNA 3 weeks after surgical sympathectomy. Protein expression also increased at this point. In addition, capillary basement membrane thickness increased significantly. NG2 proteoglycan staining decreased significantly in pericytes in the sympathectomized rat retina. Steady state mRNA for PDGF-BB decreased significantly 6 weeks after surgery. CONCLUSIONS. Sympathetic nerves may be compromised in diabetes, and these findings suggest that they may regulate some complications of diabetic retinopathy. Gene expression levels of fibronectin and laminin-␤1 changed between 1 and 3 weeks. These data are supported by electron microscopy, which showed the increase in basement membrane thickness in vivo. Loss of sympathetic innervation to the eye also caused a decrease in the number of pericytes. Steady state mRNA expression of PDGF-BB was reduced, suggesting a mechanism for the loss of pericytes in the sympathectomized retina. Overall, these results suggest that sympathetic nerve alterations may function in some complications observed in diabetic retinopathy, and this may be a suitable model to investigate therapies for this disorder. 1,2 The principal characteristics of diabetic retinopathy are thickening of the basement membrane in the retina, loss of pericytes, increased proliferation of endothelial cells, and formation of microaneurysms, which can lead to neovascularization. 3 However, it is still unclear how these alterations are caused by chronic diabetes. Alterations to sympathetic innervation in the eye could contribute to diabetes-induced change. Sympathetic nerves are significantly altered in diabetes. 6 These changes are mediated by ␤-adrenergic receptors, since administration of propranolol causes changes similar to those noted in sympathectomy. 7 Even though the retina is autoregulated, we and others To test this hypothesis, female Sprague-Dawley rats underwent surgical removal of the superior cervical ganglion, followed by assessment of gene and protein expression of two key basement membrane components (laminin-␤1 and fibronectin), electron microscopy to determine basement membrane thickness, and evaluation of the number of pericytes. Steady state mRNA expression was also assessed for the growth factor PDGF-BB. MATERIALS AND METHODS Surgical Sympathectomy Twenty-six female Sprague-Dawley rats were anesthetized intraperitoneally at postnatal day 60 with a mixture of ketamine (60 mg/kg), atropine (0.54 mg/kg), and xylazine (8 mg/kg). The right superior cervical ganglion was removed aseptically by previously described methods. 11 Right eye ptosis was used to confirm denervation, and only rats displaying good ptosis were used in the experiments. Retinal samples were taken 1, 3, and 6 weeks after sympathectomy. The contralateral or left eye served as an intra-animal control. All surgical procedures were approved by the Institutional Animal Care and Use Committee at Southern Illinois University-Carbondale and conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and NIH guidelines. RNA Isolation and Reverse Transcription RNA was isolated from retinal samples of six rats at each time point (TriReagent; Molecular Research Center, Inc., Cincinnati, OH), by using chloroform and isopropanol. RNA purity was detected by agarose gel electrophoresis, and RNA concentration was measured spectrophotometrically. Reverse transcription of 1 g RNA for cDNA synthesis was performed (Improm II Kit; Promega, Madison, WI). The reaction mixture consisted of diethyl pyrocarbonate (DEPC) water, 5ϫ reaction buffer (Improm II; Promega), 25 mM MgCl 2, 10 mM dNTP, and 20 U RNAsin. Strands were extended for 60 minutes at 42°C, and the reverse transcriptase enzyme was heat inactivated at 70°C for 15 minutes. RNase A inhibitor (0.2 L; 10 mg/mL) was added, followed by incubation for 30 minutes at 37°C. Samples were stored at Ϫ20°C for real-time PCR. From the Departments of 1 Physiology an

Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest–derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre(+);Rb1(fl/fl) mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

<p>Abstract</p> <p>Background</p> <p><it>Tetrahymena thermophila</it>, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of <it>Tetrahymena</it>'s coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.</p> <p>Results</p> <p>We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.</p> <p>Conclusion</p> <p>We report here significant progress in genome closure and reannotation of <it>Tetrahymena thermophila</it>. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.</p

Managing toxicities associated with immune checkpoint inhibitors: consensus recommendations from the Society for Immunotherapy of Cancer (SITC) Toxicity Management Working Group.

Cancer immunotherapy has transformed the treatment of cancer. However, increasing use of immune-based therapies, including the widely used class of agents known as immune checkpoint inhibitors, has exposed a discrete group of immune-related adverse events (irAEs). Many of these are driven by the same immunologic mechanisms responsible for the drugs\u27 therapeutic effects, namely blockade of inhibitory mechanisms that suppress the immune system and protect body tissues from an unconstrained acute or chronic immune response. Skin, gut, endocrine, lung and musculoskeletal irAEs are relatively common, whereas cardiovascular, hematologic, renal, neurologic and ophthalmologic irAEs occur much less frequently. The majority of irAEs are mild to moderate in severity; however, serious and occasionally life-threatening irAEs are reported in the literature, and treatment-related deaths occur in up to 2% of patients, varying by ICI. Immunotherapy-related irAEs typically have a delayed onset and prolonged duration compared to adverse events from chemotherapy, and effective management depends on early recognition and prompt intervention with immune suppression and/or immunomodulatory strategies. There is an urgent need for multidisciplinary guidance reflecting broad-based perspectives on how to recognize, report and manage organ-specific toxicities until evidence-based data are available to inform clinical decision-making. The Society for Immunotherapy of Cancer (SITC) established a multidisciplinary Toxicity Management Working Group, which met for a full-day workshop to develop recommendations to standardize management of irAEs. Here we present their consensus recommendations on managing toxicities associated with immune checkpoint inhibitor therapy

Influenza A (H3N2) Outbreak, Nepal

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain

A multi-decade record of high quality fCO2 data in version 3 of the Surface Ocean CO2 Atlas (SOCAT)

The Surface Ocean CO2 Atlas (SOCAT) is a synthesis of quality-controlled fCO2 (fugacity of carbon dioxide) values for the global surface oceans and coastal seas with regular updates. Version 3 of SOCAT has 14.7 million fCO2 values from 3646 data sets covering the years 1957 to 2014. This latest version has an additional 4.6 million fCO2 values relative to version 2 and extends the record from 2011 to 2014. Version 3 also significantly increases the data availability for 2005 to 2013. SOCAT has an average of approximately 1.2 million surface water fCO2 values per year for the years 2006 to 2012. Quality and documentation of the data has improved. A new feature is the data set quality control (QC) flag of E for data from alternative sensors and platforms. The accuracy of surface water fCO2 has been defined for all data set QC flags. Automated range checking has been carried out for all data sets during their upload into SOCAT. The upgrade of the interactive Data Set Viewer (previously known as the Cruise Data Viewer) allows better interrogation of the SOCAT data collection and rapid creation of high-quality figures for scientific presentations. Automated data upload has been launched for version 4 and will enable more frequent SOCAT releases in the future. High-profile scientific applications of SOCAT include quantification of the ocean sink for atmospheric carbon dioxide and its long-term variation, detection of ocean acidification, as well as evaluation of coupled-climate and ocean-only biogeochemical models. Users of SOCAT data products are urged to acknowledge the contribution of data providers, as stated in the SOCAT Fair Data Use Statement. This ESSD (Earth System Science Data) “living data” publication documents the methods and data sets used for the assembly of this new version of the SOCAT data collection and compares these with those used for earlier versions of the data collection (Pfeil et al., 2013; Sabine et al., 2013; Bakker et al., 2014). Individual data set files, included in the synthesis product, can be downloaded here: doi:10.1594/PANGAEA.849770. The gridded products are available here: doi:10.3334/CDIAC/OTG.SOCAT_V3_GRID

2018 Scholars at Work Conference Program

Program for the 2018 Scholars at Work Conference at Minnesota State University, Mankato on March 30, 2018

Antigenic Fingerprinting of H5N1 Avian Influenza Using Convalescent Sera and Monoclonal Antibodies Reveals Potential Vaccine and Diagnostic Targets

Using whole-genome-fragment phage display libraries, Hana Golding and colleagues identify the viral epitopes recognized by serum antibodies in humans who have recovered from infection with H5N1 avian influenza

Global carbon budget 2022

Accurate assessment of anthropogenic carbon dioxide (CO2) emissions and their redistribution among the atmosphere, ocean, and terrestrial biosphere in a changing climate is critical to better understand the global carbon cycle, support the development of climate policies, and project future climate change. Here we describe and synthesize data sets and methodologies to quantify the five major components of the global carbon budget and their uncertainties. Fossil CO2 emissions (EFOS) are based on energy statistics and cement production data, while emissions from land-use change (ELUC), mainly deforestation, are based on land use and land-use change data and bookkeeping models. Atmospheric CO2 concentration is measured directly, and its growth rate (GATM) is computed from the annual changes in concentration. The ocean CO2 sink (SOCEAN) is estimated with global ocean biogeochemistry models and observation-based data products. The terrestrial CO2 sink (SLAND) is estimated with dynamic global vegetation models. The resulting carbon budget imbalance (BIM), the difference between the estimated total emissions and the estimated changes in the atmosphere, ocean, and terrestrial biosphere, is a measure of imperfect data and understanding of the contemporary carbon cycle. All uncertainties are reported as ±1σ. For the year 2021, EFOS increased by 5.1% relative to 2020, with fossil emissions at 10.1±0.5GtCyr-1 (9.9±0.5GtCyr-1 when the cement carbonation sink is included), and ELUC was 1.1±0.7GtCyr-1, for a total anthropogenic CO2 emission (including the cement carbonation sink) of 10.9±0.8GtCyr-1 (40.0±2.9GtCO2). Also, for 2021, GATM was 5.2±0.2GtCyr-1 (2.5±0.1ppmyr-1), SOCEAN was 2.9 ±0.4GtCyr-1, and SLAND was 3.5±0.9GtCyr-1, with a BIM of -0.6GtCyr-1 (i.e. the total estimated sources were too low or sinks were too high). The global atmospheric CO2 concentration averaged over 2021 reached 414.71±0.1ppm. Preliminary data for 2022 suggest an increase in EFOS relative to 2021 of +1.0% (0.1% to 1.9%) globally and atmospheric CO2 concentration reaching 417.2ppm, more than 50% above pre-industrial levels (around 278ppm). Overall, the mean and trend in the components of the global carbon budget are consistently estimated over the period 1959-2021, but discrepancies of up to 1GtCyr-1 persist for the representation of annual to semi-decadal variability in CO2 fluxes. Comparison of estimates from multiple approaches and observations shows (1) a persistent large uncertainty in the estimate of land-use change emissions, (2) a low agreement between the different methods on the magnitude of the land CO2 flux in the northern extratropics, and (3) a discrepancy between the different methods on the strength of the ocean sink over the last decade. This living data update documents changes in the methods and data sets used in this new global carbon budget and the progress in understanding of the global carbon cycle compared with previous publications of this data set. The data presented in this work are available at 10.18160/GCP-2022 (Friedlingstein et al., 2022b)