Diabetic Müller-Glial-Cell-Specific Il6ra Knockout Mice Exhibit Accelerated Retinal Functional Decline and Thinning of the Inner Nuclear Layer.

Purpose: Interleukin-6 (IL-6) is implicated in the pathology of diabetic retinopathy (DR). IL-6 trans-signaling via soluble IL-6 receptor (IL-6R) is primarily responsible for its pro-inflammatory functions, whereas cis-signaling via membrane-bound IL-6R is anti-inflammatory. Using a Müller-glial-cell-specific Il6ra-/- mouse, we examined how loss of IL-6 cis-signaling in Müller glial cells (MGCs) affected retinal thinning and electroretinography (ERG) response over 9 months of diabetes. Methods: Diabetes was induced in wildtype and knockout mice with streptozotocin (40 mg/kg, daily for 5 days). Spectral domain optical coherence tomography (SD-OCT), ERG, and fundoscopy/fluorescein angiography (FA) were assessed at 2, 6, and 9 months of diabetes. MGCs and bipolar neurons were examined in retinal tissue sections by immunofluorescence. Results: Diabetic MGC Il6ra-/- mice had significantly thinner retinas than diabetic wildtype mice at 2 (-7.6 µm), 6 (-12.0 µm), and 9 months (-5.0 µm) of diabetes, as well as significant thinning of the inner nuclear layer (INL). Diabetic MGC Il6ra-/- mice also showed a reduction in scotopic B-wave amplitude and B-wave/A-wave ratio earlier than wildtype diabetic mice. In retinal sections, we found a decrease in bipolar neuronal marker PKCα only in diabetic MGC Il6ra-/- mice, which was significantly lower than both controls and diabetic wildtype mice. Glutamine synthetase, a Müller cell marker, was reduced in both wildtype and MGC Il6ra-/- diabetic mice compared to their respective controls. Conclusions: IL-6 cis-signaling in MGCs contributes to maintenance of the INL in diabetes, and loss of the IL-6 receptor reduces MGC-mediated neuroprotection of bipolar neurons in the diabetic retina.

Blockade of interleukin-6 trans-signaling prevents mitochondrial dysfunction and cellular senescence in retinal endothelial cells.

Interleukin-6 (IL-6) is a multifaceted cytokine implicated in the pathogenesis of diabetic retinopathy (DR). Its activity extends through cis- and trans-signaling (TS) pathways, with cis-signaling limited to specific cell types possessing the membrane-bound IL-6 receptor, while trans-signaling broadly activates various cells without the membrane bound IL-6 receptor, including retinal endothelial cells. In this study, we determined the effects of interleukin-6 trans-signaling on mitochondrial dysfunction and cellular senescence in human retinal endothelial cells (HRECs). HRECs were cultured and treated with IL-6 + soluble IL-6R or Hyper IL-6 to activate trans-signaling, along with sgp130Fc for inhibition. RT-PCR was used to analyze gene expression changes associated with inflammation and senescence. Cellular senescence was assessed using SA ß-gal staining. Mitochondrial function was evaluated using Seahorse XFe24 Bioanalyzer. IL-6 trans-signaling induced inflammatory gene expression as indicated by the upregulation of ICAM1, MCP1, and SERPINA3 levels. Additionally, it reduced mitochondrial respiration and oxidative phosphorylation, and these effects were counteracted by sgp130Fc. Moreover, IL-6 trans-signaling led to altered expression of apoptosis-associated genes, including downregulation of FIS1, BCL2, and MCL1, while promoting cellular senescence, a phenomenon mitigated by sgp130Fc. These results not only deepen our understanding of IL-6 in DR but also carry broader implications for age-related diseases and the aging process itself. This study underscores the potential therapeutic value of targeting IL-6 trans-signaling with sgp130Fc as a promising anti-inflammatory approach for DR and potentially other inflammatory conditions. Further in-vivo investigations are warranted to elucidate the function of IL-6 trans-signaling in aging-related pathologies and overall organismal health.

Generation and characterization of a Müller-glial-cell-specific Il6ra knockout mouse to delineate the effects of IL-6 trans-signaling in the retina.

Interleukin-6 (IL-6) is implicated in various retinal and vascular complications associated with diabetic retinopathy (DR). This cytokine functions through two main modalities: classical signaling, in cells expressing the membrane-bound receptor (IL-6Rα); and trans-signaling, possible in most cells through a soluble form of the receptor (sIL-6R). These pathways are considered to be anti-inflammatory and pro-inflammatory, respectively. Our recent studies in retinal endothelial cells and diabetic mice have shown that inhibiting only IL-6 trans-signaling is sufficient to prevent increased vascular leakage, oxidative stress, and inflammation characteristic of DR. Isolating the specific effects of each signaling pathway, however, remains difficult in cells expressing IL-6Rα that are thus capable of both classical and trans-signaling. Müller glial cells (MGCs), the most abundant retinal macroglial cells, span the entire retinal thickness with vital roles in maintaining retinal homeostasis and regulating the blood-retinal barrier through secreted factors. The specific effects of IL-6 trans-signaling in MGCs remain poorly understood given their responsiveness to both IL-6 signaling modalities. In this study, we addressed these concerns by generating an MGC-specific knockout mouse using Cre-loxP deletion of the Il6ra cytokine-binding region. We assessed transcriptional and translational Il6ra expression to confirm the knockout and characterized the effects of knockout on visual functioning in these mice.

A novel high-throughput sequencing approach reveals the presence of a new virus infecting Rosa: rosa ilarvirus-1 (RIV-1).

Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.

RNA-Seq analysis reveals gene expression changes induced by IL-6 trans-signaling activation in retinal endothelial cells.

BACKGROUND: Increasing evidence suggests that interleukin-6 (IL-6) trans-signaling plays a critical role in the pathogenesis of diabetic retinopathy (DR). We have previously shown that activation of IL-6 trans-signaling induces barrier dysfunction in human retinal endothelial cells (HRECs). However, the molecular mechanisms underlying these effects are not clear. The purpose of this study was to discover global gene expression changes in HRECs following activation of IL-6 trans-signaling. METHODS: HRECs were treated with IL-6 and soluble IL-6R to activate IL-6 trans-signaling, and sgp130Fc treatment was used for IL-6 trans-signaling inhibition. RNA-Seq analyses were performed for global gene expression profiling. Differential expression was determined using DESeq2, and bioinformatic analyses were performed to associate the differentially expressed genes with biological functions and pathways. RESULTS: Our analyses revealed 445 differentially expressed genes (318 upregulated and 127 downregulated) in HRECs after IL-6 trans-signaling activation. We identified several novel genes not previously associated with IL-6 signaling or endothelial dysfunction. Leukocyte adhesion, diapedesis and chemokine signaling pathways are highly enriched in differentially expressed genes. Inhibition of IL-6 trans-signaling with sgp130Fc abrogated these changes, thus highlighting the therapeutic potential of this drug. CONCLUSIONS: This study identified significant gene expression changes caused by IL-6 trans-signaling activation in HRECs. Identification of such changes has the potential to uncover the precise molecular mechanisms of IL-6 trans-signaling mediated effects in the pathology of DR.

Culture-based Methods for Studying the Bacterial Root Microbiome of Wheat.

Beneficial plant-microbe interactions are important and desirable for sustainable intensification of agriculture. Here, we describe methods to isolate microbes from the roots of field-grown wheat plants. This includes the rhizosphere and rhizoplane soil, as well as the root endosphere. We also describe a method to test for endosphere competence of putative endophytes.

Diabetes Induced Alterations in Murine Vitreous Proteome Are Mitigated by IL-6 Trans-Signaling Inhibition.

Purpose: Diabetic retinopathy (DR) is a microvascular complication caused by prolonged hyperglycemia and characterized by leaky retinal vasculature and ischemia-induced angiogenesis. Vitreous humor is a gel-like biofluid in the posterior segment of the eye between the lens and the retina. Disease-related changes are observed in the biochemical constituents of the vitreous, including proteins and macromolecules. Recently, we found that IL-6 trans-signaling plays a significant role in the vascular leakage and retinal pathology associated with DR. Therefore, in this study, comprehensive proteomic profiling of the murine vitreous was performed to identify diabetes-induced alterations and to determine effects of IL-6 trans-signaling inhibition on these changes. Methods: Vitreous samples from mice were collected by evisceration, and proteomic analyses were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: A total of 154 proteins were identified with high confidence in control mice and were considered to be characteristic of healthy murine vitreous fluid. The levels of 72 vitreous proteins were significantly altered in diabetic mice, including several members of heat shock proteins, 14-3-3 proteins, and tubulins. Alterations in 52 out of 72 proteins in diabetic mice were mitigated by IL-6 trans-signaling inhibition. Conclusions: Proteomic analysis of murine vitreous fluid performed in this study provides important information about the changes caused by diabetes in the ocular microenvironment. These diabetes-induced alterations in the murine vitreous proteome were mitigated by IL-6 trans-signaling inhibition. These findings further support that IL-6 trans-signaling may be an important therapeutic target for the treatment of DR.

Interleukin-6 trans-signaling inhibition prevents oxidative stress in a mouse model of early diabetic retinopathy.

PURPOSE: Diabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of visual disability and blindness in diabetic patients. Chronic hyperglycemia leads to increased oxidative stress and inflammation in the retina, resulting in microvascular damage. Our recent in vitro studies have demonstrated that inhibition of interleukin-6 (IL-6) trans-signaling significantly reduces oxidative stress in retinal endothelial cells. The purpose of this study was to further explore the relationship between IL-6 trans-signaling and oxidative stress using a streptozotocin (STZ) induced mouse model of early diabetic retinopathy. METHODS: Diabetes was induced in eight week-old male C57BL/6J mice using STZ injections. sgp130Fc (mouse sgp130Fc protein) treatment was used for inhibition of IL-6 trans-signaling. Studies were conducted to evaluate the effects of IL-6 trans-signaling on oxidative balance at the systemic and retinal level. RESULTS: Decreased antioxidant capacity and increased oxidative stress was observed in diabetic mice, which returned to near-normal levels with sgp130Fc treatment. Similarly, superoxide levels, lipid peroxidation, and markers of oxidative DNA damage were increased in the diabetic retina, and these effects were abrogated by sgp130Fc treatment. Inhibition of IL-6 trans-signaling also restored normal expression of catalase and endothelial nitric oxide synthase in mouse retinas. CONCLUSIONS: Inhibition of IL-6 trans-signaling significantly reduces diabetes-induced oxidative damage at the systemic level and in the retina. These findings provide further evidence for the role of IL-6 trans-signaling in diabetes-mediated oxidative stress.

Wheat dwarfing influences selection of the rhizosphere microbiome.

The development of dwarf wheat cultivars combined with high levels of agrochemical inputs during the green revolution resulted in high yielding cropping systems. However, changes in wheat cultivars were made without considering impacts on plant and soil microbe interactions. We studied the effect of these changes on root traits and on the assembly of rhizosphere bacterial communities by comparing eight wheat cultivars ranging from tall to semi-dwarf plants grown under field conditions. Wheat breeding influenced root diameter and specific root length (SRL). Rhizosphere bacterial communities from tall cultivars were distinct from those associated with semi-dwarf cultivars, with higher differential abundance of Actinobacteria, Bacteroidetes and Proteobacteria in tall cultivars, compared with a higher differential abundance of Verrucomicrobia, Planctomycetes and Acidobacteria in semi-dwarf cultivars. Predicted microbial functions were also impacted and network analysis revealed a greater level of connectedness between microbial communities in the tall cultivars relative to semi-dwarf cultivars. Taken together, results suggest that the development of semi-dwarf plants might have affected the ability of plants to recruit and sustain a complex bacterial community network in the rhizosphere.

Land Management and Microbial Seed Load Effect on Rhizosphere and Endosphere Bacterial Community Assembly in Wheat.

Microbial community ecology studies have traditionally utilized culture-based methodologies, though the advent of next-generation amplicon sequencing has facilitated superior resolution analyses of complex microbial communities. Here, we used culture-dependent and -independent approaches to explore the influence of land use as well as microbial seed load on bacterial community structure of the wheat rhizosphere and root endosphere. It was found that niche was an important factor in shaping the microbiome when using both methodological approaches, and that land use was also a discriminatory factor for the culture-independent-based method. Although culture-independent methods provide a higher resolution of analysis, it was found that in the rhizosphere, particular operational taxonomic units (OTUs) in the culture-dependent fraction were absent from the culture-independent fraction, indicating that deeper sequence analysis is required for this approach to be exhaustive. We also found that the microbial seed load defined the endosphere, but not rhizosphere, community structure for plants grown in soil which was not wheat adapted. Together, these findings increase our understanding of the importance of land management and microbial seed load in shaping the root microbiome of wheat and this knowledge will facilitate the exploitation of plant-microbe interactions for the development of novel microbial inoculants.

Proteomic Biomarkers of Retinal Inflammation in Diabetic Retinopathy.

Diabetic retinopathy (DR), a sight-threatening neurovasculopathy, is the leading cause of irreversible blindness in the developed world. DR arises as the result of prolonged hyperglycemia and is characterized by leaky retinal vasculature, retinal ischemia, retinal inflammation, angiogenesis, and neovascularization. The number of DR patients is growing with an increase in the elderly population, and therapeutic approaches are limited, therefore, new therapies to prevent retinal injury and enhance repair are a critical unmet need. Besides vascular endothelial growth factor (VEGF)-induced vascular proliferation, several other mechanisms are important in the pathogenesis of diabetic retinopathy, including vascular inflammation. Thus, combining anti-VEGF therapy with other new therapies targeting these pathophysiological pathways of DR may further optimize treatment outcomes. Technological advancements have allowed for high-throughput proteomic studies examining biofluids such as aqueous humor, vitreous humor, tear, and serum. Many DR biomarkers have been identified, especially proteins involved in retinal inflammatory processes. This review attempts to summarize the proteomic biomarkers of DR-associated retinal inflammation identified over the last several years.

Comparative STAT3-Regulated Gene Expression Profile in Renal Cell Carcinoma Subtypes.

Renal cell carcinomas (RCC) are heterogeneous and can be further classified into three major subtypes including clear cell, papillary and chromophobe. Signal transducer and activator of transcription 3 (STAT3) is commonly hyperactive in many cancers and is associated with cancer cell proliferation, invasion, migration, and angiogenesis. In renal cell carcinoma, increased STAT3 activation is associated with increased metastasis and worse survival outcomes, but clinical trials targeting the STAT3 signaling pathway have shown varying levels of success in different RCC subtypes. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we compared expression of 32 STAT3 regulated genes in 3 RCC subtypes. Our results indicate that STAT3 activation plays the most significant role in clear cell RCC relative to the other subtypes, as half of the evaluated genes were upregulated in this subtype. MMP9, BIRC5, and BCL2 were upregulated and FOS was downregulated in all three subtypes. Several genes including VEGFA, VIM, MYC, ITGB4, ICAM1, MMP1, CCND1, STMN1, TWIST1, and PIM2 had variable expression in RCC subtypes and are potential therapeutic targets for personalized medicine.

Candida albicans-derived mannoproteins activate NF-κB in reporter cells expressing TLR4, MD2 and CD14.

The ability of soluble C. albicans 20A (serotype A) mannoprotein (CMP) to serve as a ligand for toll-like receptor 4 (TLR4) and its co-receptors was examined using commercially available and stably-transfected HEK293 cells that express human TLR4, MD2 and CD14, but not MR. These TLR4 reporter cells also express an NF-κB-dependent, secreted embryonic alkaline phosphatase (SEAP) reporter gene. TLR4-reporter cells exhibited a dose-dependent SEAP response to both LPS and CMP, wherein peak activation was achieved after stimulation with 40-50 µg/mL of CMP. Incubation on polymyxin B resin had no effect on CMP's ligand activity, but neutralized LPS-spiked controls. HEK293 Null cells lacking TLR4 and possessing the same SEAP reporter failed to respond to LPS or CMP, but produced SEAP when activated with TNFα. Reporter cell NF-κB responses were accompanied by transcription of IL-8, TNFα, and COX-2 genes. Celecoxib inhibited LPS-, CMP-, and TNFα-dependent NF-κB responses; whereas, indomethacin had limited effect on LPS and CMP responses. SEAP production in response to C. albicans A9 mnn4Δ mutant CMP, lacking phosphomannosylations on N-linked glycans, was significantly greater (p ≤ 0.005) than SEAP responses to CMP derived from parental A9 (both serotype B). These data confirm that engineered human cells expressing TLR4, MD2 and CD14 can respond to CMP with NF-κB activation and the response can be influenced by variations in CMP-mannosylation. Future characterizations of CMPs from other sources and their application in this model may provide further insight into variations observed with TLR4 dependent innate immune responses targeting different C. albicans strains.

Using an FPLC to promote active learning of the principles of protein structure and purification.

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and non-native). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017.

Chimeric approach for narrowing a membrane-inserting region within human perforin.

Perforin is a pore-forming, immune protein that functions to deliver an apoptotic cocktail of proteins into a target pathogen. Recent studies of the bacterial cholesterol-dependent cytolysins (CDCs) have provided a model for perforin's pore-forming mechanism. Both perforin and CDC family members share a conserved ß-sheet flanked by two clusters of α-helices. Within the CDCs, these helices refold into two transmembrane ß-hairpins, TMH1 and TMH2. Based upon structural conservation and electron microscopy imaging, the analogous helices within perforin are predicted to also be membrane inserting; however, these regions are approximately twice the length of the CDC TMHs. To test the membrane-insertion potential of one of these regions, chimeras were created using a well-characterized CDC, perfringolysin-O (PFO), as the backbone of these constructs. PFO's TMH2 region was replaced with perforin's corresponding helical region. Although hemolytic activity was observed, the chimera was poorly soluble. A second chimera contained the same region truncated to match the length of the PFO TMH2 region. The truncated chimera demonstrated improved solubility, significant hemolytic activity and the ability to form pores characteristic of those created by PFO. These results provide the first evidence that perforin's helices function as TMHs and more importantly narrows the residues responsible for membrane insertion.

Wheat seed embryo excision enables the creation of axenic seedlings and Koch's postulates testing of putative bacterial endophytes.

Early establishment of endophytes can play a role in pathogen suppression and improve seedling development. One route for establishment of endophytes in seedlings is transmission of bacteria from the parent plant to the seedling via the seed. In wheat seeds, it is not clear whether this transmission route exists, and the identities and location of bacteria within wheat seeds are unknown. We identified bacteria in the wheat (Triticum aestivum) cv. Hereward seed environment using embryo excision to determine the location of the bacterial load. Axenic wheat seedlings obtained with this method were subsequently used to screen a putative endophyte bacterial isolate library for endophytic competency. This absence of bacteria recovered from seeds indicated low bacterial abundance and/or the presence of inhibitors. Diversity of readily culturable bacteria in seeds was low with 8 genera identified, dominated by Erwinia and Paenibacillus. We propose that anatomical restrictions in wheat limit embryo associated vertical transmission, and that bacterial load is carried in the seed coat, crease tissue and endosperm. This finding facilitates the creation of axenic wheat plants to test competency of putative endophytes and also provides a platform for endophyte competition, plant growth, and gene expression studies without an indigenous bacterial background.

A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment.