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Insects account for ~80% of the world’s species, translating to approximately 200 million insects for every human. Moths and butterflies, from the order Lepidoptera, belong to second most diverse order of insects. The Cabbage Looper, Trichoplusia ni, is a prolific crop pollinator. Unfortunately, its larvae are worldwide agricultural pests because they consume copious quantities of food, eating 3-4 times their body weight in food daily, and growing 1000-fold in just a few weeks. These invasive agricultural crop pests are generalist feeders (eating >160 beneficial host plants) and consume large quantities of food to support their rapid growth. Rapid larval growth requires specialized regulatory mechanisms and an efficient excretory system. The Malpighian tubules (MTs) function as the insects’ primary excretory organ (equivalent to the vertebrate kidney), regulating ion and fluid transport, and xenobiotic and metabolic waste excretion. MT epithelium of larval lepidopterans is of particular interest because it can rapidly (~10 min) switch between ion reabsorption (where ions are transported from tubule lumen to hemolymph) and ion secretion (where ions are transported from hemolymph to tubule lumen). Recent studies have shown that this switch has been shown to be regulated in part by voltage-gated ion channels (VGICs). In larval T. ni, CaV1 channels expressed in the MTs distal ileac plexus region (DIP) have been shown to directly regulate K+ secretion. Pharmacological inhibition of voltage-gated ion channels (Kir1, HCN1, Cav1) independently from each other altered cell-based K+ transport. In the current study, biological assays measured changes in fluid secretion rate and membrane potential of MT epithelia of larval lepidopterans in response to pharmacological inhibition and/or activation of VGICs. In addition, immunohistochemistry assay using custom-made antibodies, revealed cellular and membrane localization of voltage-gated TRPA/ painless in the epithelia of the MT’s. In the current study we demonstrate that activating and inhibiting voltage-gated K+ and Ca2+ channels altered fluid secretion rate and membrane potential in the MTs of larval lepidopterans. VGICs have been extensively studied in excitable tissues. In contrast, their roles in the regulation of ion transport in the non-contractile, non-innervated epithelia of animals remains largely understudied. The novel data obtained in this study provides further insight about the fast-acting response mechanisms of VGICs, and how they play a vital role in the regulation of ion and water transp California State University startup and seed grant funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HEAT UNIT REQUIREMENT IN RELATION TO PHENOLOGY OF CHICKPEA CULTIVARS AS INFLUENCED BY SOWING DATES

Effect of different sowing dates on crop phenology and seed yield attributes in Indian mustard genotypes

EFFECT OF DATES OF TRANSPLANTING ON APHIDS INFESTING CABBAGE, Brassica oleracea var. capitata. L.

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

Adhesion G-Protein Coupled Receptors (aGPCRs) have evolved an activation mechanism to translate extracellular force into liberation of a tethered agonist (TA) to modulate cell signalling. We report here that ADGRF1 is the first class B GPCR shown to signal through all major G-protein classes and identify the structural basis for its Gαq preference by cryo-EM. Our structure shows that Gαq over Gαs preference in ADGRF1 derives from tighter packing at the conserved F569 of the TA, altering contacts between TM helix I and VII, with a concurrent rearrangement of TM helices VII and VIII at the site of Gα recruitment. Gαs signalling is also more sensitive to mutation of TA or binding site residues than Gαq. Our work advances the understanding of aGPCR TA activation in molecular detail, identifying structural features that potentially explain preferential signal modulation.

In this study, we will evaluate classroom performance and how it is affected by the amount of non-academic technology use during lectures.

Adhesion G Protein-Coupled Receptors (aGPCRs) are a distinct family of transmembrane proteins that carry out numerous cellular functions in multicellular organisms. During maturation, aGPCRs undergo autoproteolysis to generate non-covalently associated N-terminal and C-terminal fragments (NTF and CTF, respectively). Signaling is induced when a tethered agonist at the N-terminus of the CTF is released from or unmasked by the NTF to access its binding site within the seven transmembrane domain of the CTF. Here, we show that the tethered sequence does not require a free N-terminus to signal and exploit this finding to create a fusion protein platform for interrogation of structure-function relationships in aGPCRs. We analyze tethered agonism in CD97, a protein implicated in immune cell function and cancer cell invasiveness, and demonstrate the platform’s generality by creating constitutively active forms of several other aGPCRs. This platform should find wide applicability in analyzing signals transmitted by tethered agonists in aGPCRs.

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

Cancer is considered one of the leading causes of death in the United States. Although preventive strategies, early detection, and improved treatment options have been developed, novel targets and therapeutics are still needed. Since concluding that cancer is mediated by genetic and epigenetic alterations of the cell, many research groups are now focusing on other means of prevention and therapy via nutrition, epigenetic mechanisms, and non-coding RNAs which have been shown to control gene expression and have many different functions at the cellular level. With the advent of high-throughput sequencing in human cancer, the potential to identify novel biomarkers and therapeutic targets of disease has increased tremendously and led to the identification of many non-coding RNAs that are dysregulated in various cancers. Gene expression and regulation is important in maintaining the homeostasis of normal tissues and cells. Not uncommonly, up- or down-regulation of particular genes are associated with cancer as a result of increased or decreased expression of transcriptional targets. This review focuses on the role of nutrition in cancer and the dysregulation of non-coding RNAs with particular emphasis on long non-coding RNAs and microRNAs in different cancer types.

Optimity Advisors conducted the study “Health system performance assessment – Integrated Care Assessment”, commissioned by Chafea and DG SANTE, assessing: (i) the state of integrated care (IC) implementation in the EU28 Member States, Norway and Iceland; and (ii) developing an evidence-based Integrated Care Performance Assessment (ICPA) framework. The framework, supported by an Excel tool, was co-designed with stakeholders across Europe. The ICPA tool will be tested by members of the European Innovation Partnership on Active and Healthy Ageing (EIPonAHA), B3 Action Group on Integrated Care. From a review of literature in 24 languages, an extensive mapping of 560 IC initiatives in 30 countries and the maturity assessment of 12 health systems, we inferred that IC strategies, policies, models and projects are present across Europe, but their characteristics, depth and breadth of integration vary considerably. Taking this heterogeneity into account, the framework was co-designed drawing on user-centred design and agile methodologies. Following an iterative approach with regular stakeholder engagement (questionnaires, video-conferences, peer-review webinars, presentations at events, and a validation workshop), experts and practitioners helped us determine which indicators sourced from existing performance assessment frameworks were considered “core” to IC assessment, with the rest proposed as “optional” indicators in the performance assessment model. The core indicators selected were categorised under four domains -Advancement of integration; Use of care services; Health outcomes; Experiences of care- with a fifth overarching domain considering financial issues. Indicators have been re-worded and re-defined to make them applicable across the wealth of IC initiatives while offering measures for international comparison. The heterogeneity of the context in which IC initiatives operate in Europe was a key challenge for the development of a framework that had to be relevant and potentially used in all included countries. This was addressed by providing an additional Excel-based ICPA tool offering flexibility in the use of the framework and allowing users to adapt indicators to their context. The tool allows users to compare performance to a set target for each indicator in the ICPA framework. The use of the ICPA framework and tool will help healthcare systems across Europe to further develop their IC system - based on their current state and context - and could significantly help them achieve better health outcomes and care experiences. The use of user-centred design and agile methodologies are proved methods of finding useful solutions to common challenges. Our research indicates that these principles also apply in the area of health performance assessment and can support IC implementation. Without user engagement, there was a risk that the output of the study would not provide the value sought. As IC evolves, the framework will need to be updated and adapted to new circumstances. Further user engagement and testing is recommended to ensure usability. With the research concluded, the framework and tool will need to be tested in practice through the involvement of members of the B3 Action Group of the EIPonAHA. Preliminary results will be resented at ICIC19. A web-based tool could facilitate uptake and data collection and analysis.

Alternative splicing (AS), a tightly regulated process, contributes to proteome diversification essential for tissue development and identity. During brain development, splicing targets include genes involved in several cellular processes like endocytosis, cytoskeleton dynamics and vesicle-mediated transport. Deregulation of AS in cancers enables tumor cells to alter the transcriptome in favor of isoforms that drive tumor progression. Annexin A7 (ANXA7) is a hydrophilic protein that binds membranes in a calcium-dependent manner; it is important for membrane scaffolding and vesicle trafficking. ANXA7 is alternatively spliced by PTBP1 and expressed as two isoforms - unspliced ANXA7 Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). We determined that this splicing of ANXA7 is lineage-specific in the brain - I1 is highly expressed in post-mitotic mature neurons while I2 is expressed in neural and glial precursors. In order to understand the functional impact of these isoforms, we generated GBM cell lines that express endogenous I2 (I2 cells), or endogenous I2 + exogenous I1 (I1 cells). We evaluated the influence of I1 and/or I2 expression on RTK levels, activation and downstream events using western blot and co-immunoprecipitation assays. Receptor trafficking was observed using flow cytometry and immunofluorescence assays tagging different components of the endocytic pathway. In GBM, we observed high levels of PTBP1 with a subsequent increase in I2 levels indicative of dysregulated AS. Consequently, we observed upregulated activation of several receptor tyrosine kinases (RTKs) such as EGFR, MET, PDGFRα and EGFR vIII in the I2 cells. In I1 cells, however, we observed reduced levels and activation of all aforementioned RTKs along with the diminished activation of downstream pathways. Using EGFR signaling as a model, we found that in I1 cells EGFR co-localized with markers of lysosomal degradation indicating efficient trafficking of activated EGFR for endosomal degradation; co-localization was absent in I2 cells indicative of impaired trafficking. We propose that I1, by virtue of the amino acids encoded by cassette exon 6, binds to conserved motifs within multiple RTKs and targets them for endosomal degradation thereby attenuating tumorigenic signaling in GBM. Our results show that ANXA7 I1 is tumor suppressive in GBM and that loss of I1 promotes tumorigenicity by perpetuating RTK signaling. We present a mechanism that explains, in part, how I1 mediates the degradation of multiple tumorigenic RTKs by spatio-temporally regulating their endosomal trafficking. A refined understanding of how I1 functions will provide the foundation for future, selective therapies that target dysregulated AS in GBM. Citation Format: Sindhu Nair, Rajani Rajbhandari, Centdrika Dates-Hurt, Markus Bredel. Dysregulated splicing of Annexin A7 impairs receptor tyrosine kinase trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3747.

Purpose/Objective(s): Glioblastomas (GBM) are highly malignant tumors that arise from astrocytes. GBMs rely on signaling through receptor tyrosine kinases (RTK) to ensure tumorigenicity. EGFR, PDGFRA and MET are distinct RTKs; each is capable of binding a ligand, which promotes receptor dimerization and kinase activation. Annexin A7 (ANXA7), a protein involved in membrane binding and vesicle trafficking, is spliced into 2 isoforms - isoforms 1 (I1) and isoform 2 (I2). A normal adult brain will express both isoforms, however, in GBM, I1 expression is prevented. We determined that I1, but not I2, is tumor suppressive, in part, because it promotes the endocytic degradation of EGFR. Because I1 is typically not expressed in GBM, EGFR signaling persists unabated and tumors thrive. Recently, we extended these observations and determined that I1 also impacts additional RTKs, including PDGFRA and MET. By understanding how ANXA7 I1 impacts PDGFRA and MET signaling, we hope to target multiple tumorigenic RTKs and improve therapy for patients with GBM. Materials/Methods: To understand how and when I1 downregulates PDGFRα and MET, U251-malignant glioma (MG) cells, which only express I2 (P), were modified to express I1. Cells were serum starved overnight, and then stimulated with PDGFAA and HGF(20 ng/ml), which activate the aforementioned receptors respectively. As a positive control, cells were stimulated with EGF to activate EGFR. Total protein was then collected and analyzed by western blot analyses. Next, we sought to determine whether I1 was competent to down-regulate several RTKs coincidentally. To do this, P and I1 cells were serum starved, stimulated with EGF, PDGFAA or both, and total protein levels were evaluated. Results: The levels of activated EGFR, PDGFRα and MET are dramatically reduced in those cells that express I1 as compared to cells expressing I2 (P). Upon activation with both PDGFAA and EGF, in cells expressing I1 (I1), levels of PDGFRα and EGFR were markedly reduced 30 minutes post-stimulation as compared to cells expressing I2. Conclusion: Our results suggest I1 may be a master regulator of RTK endocytosis and degradation. Further investigations of the impact of I1 on endocytic trafficking patterns using immunofluorescence and confocal microscopy are underway. Collectively, understanding how I1 functions and targets multiple tumorigenic RTKs will provide the foundation for future selective therapies that restore I1 expression in GBM and reduce tumorigenicity. Citation Format: Zachary B. White, Centdrika Dates Hurt, Rajani Rajbhandari, Sindhu Nair, Susan Nozell, Markus Bredel. Impact of ANXA7 I1 expression on PDGFRA and MET endosomal trafficking in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3758.

Dysregulation of receptor tyrosine kinase genes is a frequent genetic abnormality in GBM. A common genetic alteration is amplification and/or overexpression of the epidermal growth factor receptor (EGFR) gene. Annexin A7 (ANXA7) is a hydrophilic protein that binds membranes in a calcium dependent manner; it is important for membrane scaffolding and vesicle trafficking. ANXA7 is alternatively spliced and expressed as two isoforms – unspliced ANXA7 Isoform 1 (I1), containing cassette exon 6 and spliced Isoform 2 (I2). Our lab has determined that I1 is tumor suppressive in GBM and that loss of I1 promotes tumorigenicity by perpetuating EGFR signaling. However, if restored, I1 but not I2, reduces EGFR levels, activation and downstream pathways and diminishes tumorigenicity. In patients, I1 levels directly correlate with survival and prognosis. We propose that I1 inhibits EGFR signalling by binding to and promoting degradation of EGFR in a calcium dependent manner. In GBM, I1 is absent, preventing endosomal degradation of EGFR and leading to perpetual signalling. Our approach is novel; rather than address how/whether EGFR is activated, we focus on why EGFR fails to be downregulated afterwards. Herein, we present a mechanism that explains, in part, how I1 mediates endosomal degradation of EGFR. Further studies are underway to identify exactly where and how I1 modifies endosomal trafficking and degradation of EGFR. We are also exploring the role of I1 in regulating the degradation of other receptor tyrosine kinases (RTKs), such as MET and PDGFRA. Collectively, understanding how I1 functions will provide the foundation for future, selective therapies that restore I1 expression and/or function to reduce GBM tumorigenicity.

Background: Funded by the Third EU Public Health Programme, Optimity Advisors has followed up on the work carried out by the Health System Performance Assessment HSPA Expert Group in the development of an assessment framework for integrated care. The study conducted has assessed the level penetration and adoption of integrated care in the EU28, Norway and Iceland, and the level of maturity of several health systems at national, regional and local level. Through engagement with stakeholders in the integrated care field, a new, user-friendly framework for the performance assessment of integrated care has been co-developed and further validated by Optimity Advisors, DG SANTE i.e. Directorate-General for Health and Consumers of the European Union, CHAFEA Consumers, Health, Agriculture and Food Executive Agency and stakeholders across the integrated care community in the 28 Member States, Norway and Iceland. The Integrated Care Performance Assessment Framework was developed based on previously validated frameworks, such as the WHO 2015 framework People-centred and integrated health services: an overview of the evidence. Aims and Objectives: The main objective of this workshop will be to communicate the preliminary results and the experience of using the framework to assess the performance of a number of integrated care systems at local, regional and national level. Format: The agenda for the workshop will be as follows: Introduction to the methodological approach and preliminary outputs of the framework, by Optimity Advisors and DG SANTE; Presentation by one or two representatitves of integrated care projects who have contributed in the co-design of the framework explaining the design process; Demonstration of the use of the framework by representatitves of integrated care projects, who will show how to obtain a performance profile for integrated care systems; Group discussion on the different conditions in different health systems that can enhance, or limit the adoption of the framework, and on what could be done to minimise the barriers and maximise facilitating conditions; Conclusion by the facilitators on future steps to address the issues raised by the attendees, and final remarks. Target audience: We envision the target audience of the workshop to be comprised of individuals who have an integrated care performance assessment need, including health managers, policy makers, and other stakeholders that are directly involved in decision-making actions within integrated health systems. Learnings/Take away: By the end of the session, we expect attendees to i have an understanding of a new way to assess the performance of integrated care systems, ii develop a performance profile and actionable roadmap for performance improvement of an integrated care system of their choosing.