Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids.

Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11.

What do spouse primary caregivers of patients with glioblastoma want medical providers to know? A qualitative thematic reflexive analysis of letters written by primary caregivers from a secret Facebook support group.

OBJECTIVES: To analyse the content of letters written by female spouse primary caregivers of patients with glioblastoma multiforme (GBM), a devastating and terminal primary brain cancer, and give voice to their experiences for medical providers of patients with GBM. DESIGN: A qualitative study using reflexive thematic analysis of letters written by female spouses/life partners and primary caregivers of patients with GBM. PARTICIPANTS: 101 current or former female spouse primary caregivers of patients with GBM wrote letters to share with the medical community between July 2019 and August 2019. INCLUSION CRITERIA: (1) the primary caregiver who is a spouse of a patient with glioblastoma, (2) be a member of the secret Facebook group, 'We are the wives of GBM and this is our story', and (3) completed informed consent for the contents of their letter to be included for primary and secondary data analysis. Participants who wrote letters but did not complete the informed consent were excluded from the study. RESULTS: Themes from the letters included the patient experiences: (1) medical details of the disease trajectory, (2) interactions of the patient/caregiver dyads with healthcare and (3) the changing patient condition over time. Themes focused on the caregiver experiences: (1) caregiver challenges, (2) caregiver responses and (3) caregiver coping strategies, and description of tangible needs that would help other caregivers in the future. Caregiver needs were highest during the living with disease progression phase. Caregivers wanted more education and to be valued as members of the care team. CONCLUSION: Shared decision-making through family-centred care would be beneficial for primary caregivers of patients with GBM. These findings provide opportunities to guide more timely and tailored interventions to provide support and improve care for patient/caregiver dyads to help mitigate the burden of this progressive disease and improve quality of life for caregivers.

Bi-directional signaling between the intestinal epithelium and type-3 innate lymphoid cells regulates secretory dynamics and interleukin-22.

Type-3 innate lymphoid cells (ILC3) respond to localized environmental cues to regulate homeostasis and orchestrate immunity in the intestine. The intestinal epithelium is an important upstream regulator and downstream target of ILC3 signaling, however, the complexity of mucosal tissues can hinder efforts to define specific interactions between these two compartments. Here, we employ a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3 to uncover bi-directional signaling mechanisms that underlie intestinal homeostasis. We report that ILC3 induce global transcriptional changes in intestinal epithelial cells, driving the enrichment of secretory goblet cell signatures. We find that SIO enriched for goblet cells promote NKp46+ ILC3 and interleukin (IL)-22 expression, which can feedback to induce IL-22-mediated epithelial transcriptional signatures. However, we show that epithelial regulation of ILC3 in this system is contact-dependent and demonstrate a role for epithelial Delta-Like-Canonical-Notch-Ligand (Dll) in driving IL-22 production by ILC3, via subset-specific Notch1-mediated activation of T-bet+ ILC3. Finally, by interfering with Notch ligand-receptor dynamics, ILC3 appear to upregulate epithelial Atoh1 to skew secretory lineage determination in SIO-ILC3 co-cultures. This research outlines two complimentary bi-directional signaling modules between the intestinal epithelium and ILC3, which may be relevant in intestinal homeostasis and disease.

Characterization of the GGPP synthase gene family in Arabidopsis thaliana.

Geranylgeranyl diphosphate (GGPP) is a key precursor of various isoprenoids that have diverse functions in plant metabolism and development. The annotation of the Arabidopsis thaliana genome predicts 12 genes to encode geranylgeranyl diphosphate synthases (GGPPS). In this study we analyzed GGPPS activity as well as the subcellular localization and tissue-specific expression of the entire protein family in A. thaliana. GGPPS2 (At2g18620), GGPPS3 (At2g18640), GGPPS6 (At3g14530), GGPPS7 (At3g14550), GGPPS8 (At3g20160), GGPPS9 (At3g29430), GGPPS10 (At3g32040) and GGPPS11 (At4g36810) showed GGPPS activity in Escherichia coli, similar to activities reported earlier for GGPPS1 (At1g49530) and GGPPS4 (At2g23800) (Zhu et al. in Plant Cell Physiol 38(3):357-361, 1997a; Plant Mol Biol 35(3):331-341, b). GGPPS12 (At4g38460) did not produce GGPP in E. coli. Based on DNA sequence analysis we propose that GGPPS5 (At3g14510) is a pseudogene. GGPPS-GFP (green fluorescent protein) fusion proteins of the ten functional GGPP synthases localized to plastids, mitochondria and the endoplasmic reticulum, with the majority of the enzymes located in plastids. Gene expression analysis using quantitative real time-PCR, GGPPS promoter-GUS (ß-glucuronidase) assays and publicly available microarray data revealed a differential spatio-temporal expression of GGPPS genes. The results suggest that plastids and mitochondria are key subcellular compartments for the synthesis of ubiquitous GGPP-derived isoprenoid species. GGPPS11 and GGPPS1 are the major isozymes responsible for their biosynthesis. All remaining paralogs, encoding six plastidial isozymes and two cytosolic isozymes, were expressed in specific tissues and/or at specific developmental stages, suggesting their role in developmentally regulated isoprenoid biosynthesis. Our results show that of the 12 predicted GGPPS encoded in the A. thaliana genome 10 are functional proteins that can synthesize GGPP. Their specific subcellular location and differential expression pattern suggest subfunctionalization in providing GGPP to specific tissues, developmental stages, or metabolic pathways.

Measuring the observer (Hawthorne) effect on adenoma detection rates.

Background and study aims An independent observer can improve procedural quality. We evaluated the impact of the observer (Hawthorne effect) on important quality metrics during colonoscopies. Patients and Methods In a single-center comparative study, consecutive patients undergoing routine screening or diagnostic colonoscopy were prospectively enrolled. In the index group, all procedural steps and quality metrics were observed and documented, and the procedure was video recorded by an independent research assistant. In the reference group, colonoscopies were performed without independent observation. Colonoscopy quality metrics such as polyp, adenoma, serrated lesions, and advanced adenoma detection rates (PDR, ADR, SLDR, AADR) were compared. The probabilities of increased quality metrics were evaluated through regression analyses weighted by the inversed probability of observation during the procedure. Results We included 327 index individuals and 360 referents in the final analyses. The index group had significantly higher PDRs (62.4% vs. 53.1%, P =0.02) and ADRs (39.4% vs. 28.3%, P =0.002) compared with the reference group. The SLDR and AADR were not significantly increased. After adjusting for potential confounders, the ADR and SLDR were 50% (relative risk [RR] 1.51; 95%, CI 1.05-2.17) and more than twofold (RR 2.17; 95%, CI 1.05-4.47) more likely to be higher in the index group than in the reference group. Conclusions The presence of an independent observer documenting colonoscopy quality metrics and video recording the colonoscopy resulted in a significant increase in ADR and other quality metrics. The Hawthorne effect should be considered an alternative strategy to advanced devices to improve colonoscopy quality in practice.

Empirical Modeling of COVID-19 Evolution with High/Direct Impact on Public Health and Risk Assessment.

This report develops a conceivable mathematical model for the transmission and spread of COVID-19 in Romania. Understanding the early spread dynamics of the infection and evaluating the effectiveness of control measures in the first wave of infection is crucial for assessing and evaluating the potential for sustained transmission occurring in the second wave. The main aim of the study was to emphasize the impact of control measures and the rate of case detection in slowing the spread of the disease. Non pharmaceutical control interventions include government actions, public reactions, and other measures. The methodology consists of an empirical model, taking into consideration the generic framework of the Stockholm Environment Institute (SEI) Epidemic-Macroeconomic Model, and incorporates the effect of interventions through a multivalued parameter, a stepwise constant varying during different phases of the interventions designed to capture their impact on the model. The model is mathematically consistent and presents various simulation results using best-estimated parameter values. The model can be easily updated later in response to real-world alterations, for example, the easing of restrictions. We hope that our simulation results may guide local authorities to make timely, correct decisions for public health and risk assessment.

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

Helper-Like Type-1 Innate Lymphoid Cells in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is an idiopathic condition characterized by chronic relapsing inflammation in the intestine. While the precise etiology of IBD remains unknown, genetics, the gut microbiome, environmental factors, and the immune system have all been shown to contribute to the disease pathophysiology. In recent years, attention has shifted towards the role that innate lymphoid cells (ILCs) may play in the dysregulation of intestinal immunity observed in IBD. ILCs are a group of heterogenous immune cells which can be found at mucosal barriers. They act as critical mediators of the regulation of intestinal homeostasis and the orchestration of its inflammatory response. Despite helper-like type 1 ILCs (ILC1s) constituting a particularly rare ILC population in the intestine, recent work has suggested that an accumulation of intestinal ILC1s in individuals with IBD may act to exacerbate its pathology. In this review, we summarize existing knowledge on helper-like ILC1 plasticity and their classification in murine and human settings. Moreover, we discuss what is currently understood about the roles that ILC1s may play in the progression of IBD pathogenesis.

Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted. These systems offer versatile and reductionist models to dissect the impact of environmental and mucosal niche cues on ILC maturation. In the future, these could provide insight into how ILC activity and development might become dysregulated in chronic inflammatory diseases.

Renal Cell Carcinoma With Cardiac Metastases.

The median survival of metastatic renal cell carcinoma (mRCC) is 5 months with a 1-year survival rate of 29%. Cardiac metastasis from RCC is a rare finding and there is scarce data available on treatment options. Recently, the combination of nivolumab and ipilimumab has been approved as a first-line treatment for advanced RCC in patients with a poor prognosis. Here we present a case of a 45-year-old male who presented to the emergency room with cough, dyspnea, and fever. Chest X-ray showed hilar lymphadenopathy and diffuse reticulonodular opacities, whereas a thoracic computed tomography (CT) scan revealed carcinomatosis lymphangitis, pleural carcinosis and multiple heterogenous zones on the cardiac wall. A transthoracic echocardiogram and a cardiac magnetic resonance imaging (MRI) revealed cardiac metastases. Subsequent imaging showed abundant distal metastases whereas a renal biopsy confirmed clear cell RCC making it a high-grade stage IV metastatic RCC. The patient was treated with the combination of nivolumab and ipilimumab. The unique feature about this case is that we have found a rare case of cardiac metastases that persists after a 3-month follow-up. Previously, there was only one case report of a patient with RCC and cardiac metastases who showed persistent response to nivolumab after 12 months. The key points from this case report are that a high index of suspicion is required for diagnosing cardiac metastases given that the signs and symptoms of metastatic cardiac involvement can be non-specific. Spread has been described as directly through the renal vein and vena cava or indirectly via the lymphatic system, which confers a worse prognosis. Furthermore, cardiac metastases can be mistaken for thrombi, endocarditis, or primary tumors, therefore echocardiograms can be limiting. Supplemental imaging with cardiac MRI or positron emission tomography/CT (PET/CT) is often needed for further characterization.

Parallel analysis of Arabidopsis circadian clock mutants reveals different scales of transcriptome and proteome regulation.

The circadian clock regulates physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have integrated RNA-sequencing and protein mass spectrometry data to comparatively analyse the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette at the end of day and end of night. Each mutant affects specific sets of genes and proteins, suggesting that the circadian clock regulation is modular. Furthermore, each circadian clock mutant maintains its own dynamically fluctuating transcriptome and proteome profile specific to subcellular compartments. Most of the measured protein levels do not correlate with changes in their corresponding transcripts. Transcripts and proteins that have coordinated changes in abundance are enriched for carbohydrate- and cold-responsive genes. Transcriptome changes in all four circadian clock mutants also affect genes encoding starch degradation enzymes, transcription factors and protein kinases. The comprehensive transcriptome and proteome datasets demonstrate that future system-driven research of the circadian clock requires multi-level experimental approaches. Our work also shows that further work is needed to elucidate the roles of post-translational modifications and protein degradation in the regulation of clock-related processes.

A flexible protocol for targeted gene co-expression network analysis.

The inference of gene co-expression networks is a valuable resource for novel hypotheses in experimental research. Routine high-throughput microarray transcript profiling experiments and the rapid development of next-generation sequencing (NGS) technologies generate a large amount of publicly available data, enabling in silico reconstruction of regulatory networks. Analysis of the transcriptome under various experimental conditions proved that genes with an overall similar expression pattern often have similar functions. Consistently, genes involved in the same metabolic pathway are found in co-expressed modules. In this chapter, we describe a detailed workflow for analyzing gene co-expression networks using large-scale gene expression data and explain critical steps from design and data analysis to prediction of functionally related modules. This protocol is platform independent and can be used for data generated by ATH1 arrays, tiling arrays, or RNA sequencing for any organism. The most important feature of this workflow is that it can infer statistically significant gene co-expression networks for any number of genes and transcriptome data sets and it does not involve any particular hardware requirements.

Distinct evolutionary strategies in the GGPPS family from plants.

Multiple geranylgeranyl diphosphate synthases (GGPPS) for biosynthesis of geranylgeranyl diphosphate (GGPP) exist in plants. GGPP is produced in the isoprenoid pathway and is a central precursor for various primary and specialized plant metabolites. Therefore, its biosynthesis is an essential regulatory point in the isoprenoid pathway. We selected 119 GGPPSs from 48 species representing all major plant lineages, based on stringent homology criteria. After the diversification of land plants, the number of GGPPS paralogs per species increases. Already in the moss Physcomitrella patens, GGPPS appears to be encoded by multiple paralogous genes. In gymnosperms, neofunctionalization of GGPPS may have enabled optimized biosynthesis of primary and specialized metabolites. Notably, lineage-specific expansion of GGPPS occurred in land plants. As a representative species we focused here on Arabidopsis thaliana, which retained the highest number of GGPPS paralogs (twelve) among the 48 species we considered in this study. Our results show that the A. thaliana GGPPS gene family is an example of evolution involving neo- and subfunctionalization as well as pseudogenization. We propose subfunctionalization as one of the main mechanisms allowing the maintenance of multiple GGPPS paralogs in A. thaliana genome. Accordingly, the changes in the expression patterns of the GGPPS paralogs occurring after gene duplication led to developmental and/or condition specific functional evolution.

Network analysis of the MVA and MEP pathways for isoprenoid synthesis.

Isoprenoid biosynthesis is essential for all living organisms, and isoprenoids are also of industrial and agricultural interest. All isoprenoids are derived from prenyl diphosphate (prenyl-PP) precursors. Unlike isoprenoid biosynthesis in other living organisms, prenyl-PP, as the precursor of all isoprenoids in plants, is synthesized by two independent pathways: the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. This review focuses on progress in our understanding of how the precursors for isoprenoid biosynthesis are synthesized in the two subcellular compartments, how the underlying pathway gene networks are organized and regulated, and how network perturbations impact each pathway and plant development. Because of the wealth of data on isoprenoid biosynthesis, we emphasize research in Arabidopsis thaliana and compare the synthesis of isoprenoid precursor molecules in this model plant with their synthesis in other prokaryotic and eukaryotic organisms.

Transcript profiling in Arabidopsis with genome tiling microarrays.

Microarray technology is at present a standardized workflow for genome-wide expression analysis. Whole-genome tiling microarrays have emerged as an important platform for flexible and comprehensive expression profiling. In this chapter we describe a detailed standardized workflow for experiments assessing the transcriptome of Arabidopsis using tiling arrays and provide useful hints for critical steps from experimental design to data analysis. Although the protocol is optimized for AGRONOMICS1 arrays, it can readily be adapted to other tiling arrays. AGRONOMICS1 is the first platform that enables strand-specific expression analysis of the Arabidopsis genome with a single array. Moreover, it includes all perfect match probes from the original ATH1 array, allowing readily integration with the large existing ATH1 knowledge base. This workflow is designed for the analysis of raw data for any number of samples and it does not pose any particular hardware requirements.

Structure and dynamics of the isoprenoid pathway network.

Isoprenoids are functionally and structurally the most diverse group of plant metabolites reported to date. They can function as primary metabolites, participating in essential plant cellular processes, and as secondary metabolites, of which many have substantial commercial, pharmacological, and agricultural value. Isoprenoid end products participate in plants in a wide range of physiological processes acting in them both synergistically, such as chlorophyll and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination. It is therefore expected that fluxes via isoprenoid metabolic network are tightly controlled both temporally and spatially, and that this control occurs at different levels of regulation and in an orchestrated manner over the entire isoprenoid metabolic network. In this review, we summarize our current knowledge of the topology of the plant isoprenoid pathway network and its regulation at the gene expression level following diverse stimuli. We conclude by discussing agronomical and biotechnological applications emerging from the plant isoprenoid metabolism and provide an outlook on future directions in the systems analysis of the plant isoprenoid pathway network.