Mount Desert Island Biological Laboratory

Podocytes are kidney glomerular cells that depend on rigorously regulated cytoskeleton components and integrins to form and maintain the so-called foot processes, apparatuses that attach podocytes to the glomerular basement membrane and connect them to neighboring podocytes. In diabetic kidney disease (DKD) these foot processes are effaced as a result of cytoskeleton dysregulation, a phenomenon that gradually reduces glomerular filtration. Cytoskeleton-associated protein 4 (CKAP4) is a known linker between the endoplasmic reticulum, integrins, and microtubular cytoskeleton. Since CKAP4 gene expression is downregulated in glomeruli from patients with DKD but not in other chronic kidney diseases, we hypothesized a role for CKAP4 in the mechanisms leading to foot process effacement (FPE) in DKD. CKAP4 mRNA reduction in podocytes in DKD was demonstrated in human kidney biopsies. Knockdown of CKAP4 in vivo in zebrafish resulted in edema, proteinuria, and foot process effacement, all typical features of DKD. Knockdown of CKAP4 in vitro led to disruption of the actin cytoskeleton and of the microtubular orientation. Moreover, it caused a downregulation of several integrins. These findings indicate that CKAP4 is crucial for foot process dynamics of podocytes. Its reduction, unique to DKD, is mechanistically connected to the pathophysiological processes leading to podocyte FPE.

The formation of functional epithelial tubules is critical for the development and maintenance of many organ systems. While the mechanisms of tubule formation by epithelial cells are well studied, the process of tubule anastomosis-where tubules connect to form a continuous network-remains poorly understood. In this study, we utilized single-cell RNA sequencing to analyze embryonic mouse kidney tubules undergoing anastomosis. Our analysis identified hepatocyte growth factor (HGF) as a key potential mediator of this process. To investigate this further, we developed an assay using epithelial spheroids with fluorescently tagged apical surfaces, allowing us to visualize and quantify tubule-tubule connections. Our results demonstrate that HGF promotes tubule anastomosis, and it does so through the MAPK signaling pathway and MMPs, independently of cell proliferation. Remarkably, treatment with HGF and collagenase was sufficient to induce tubule anastomosis in embryonic mouse kidneys. These findings provide a foundational understanding of how to enhance the formation of functional tubular networks. This has significant clinical implications for the use of in vitro-grown kidney tissues in transplant medicine, potentially improving the success and integration of transplanted tissues.

This manuscript describes the development of a resource module that is part of a learning platform named “NIGMS Sandbox for Cloud-based Learning” https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on RNA sequencing (RNAseq) data analysis in an interactive format that uses appropriate cloud resources for data access and analyses. Biomedical research is increasingly data-driven, and dependent upon data management and analysis methods that facilitate rigorous, robust, and reproducible research. Cloud-based computing resources provide opportunities to broaden the application of bioinformatics and data science in research. Two obstacles for researchers, particularly those at small institutions, are: (i) access to bioinformatics analysis environments tailored to their research; and (ii) training in how to use Cloud-based computing resources. We developed five reusable tutorials for bulk RNAseq data analysis to address these obstacles. Using Jupyter notebooks run on the Google Cloud Platform, the tutorials guide the user through a workflow featuring an RNAseq dataset from a study of prophage altered drug resistance in Mycobacterium chelonae. The first tutorial uses a subset of the data so users can learn analysis steps rapidly, and the second uses the entire dataset. Next, a tutorial demonstrates how to analyze the read count data to generate lists of differentially expressed genes using R/DESeq2. Additional tutorials generate read counts using the Snakemake workflow manager and Nextflow with Google Batch. All tutorials are open-source and can be used as templates for other analysis.

The ability to maintain muscle function decreases with age and loss of proteostatic function. Diet, drugs, and genetic interventions that restrict nutrients or nutrient signaling help preserve long-term muscle function and slow age-related decline. Previously, it was shown that attenuating protein synthesis downstream of the mechanistic target of rapamycin (mTOR) gradually increases expression of heat shock response (HSR) genes in a manner that correlates with increased resilience to protein unfolding stress. Here, we investigate the role of specific tissues in mediating the cytoprotective effects of low translation. This study uses genetic tools (transgenic C. elegans , RNA interference and gene expression analysis) as well as physiological assays (survival and paralysis assays) in order to better understand how specific tissues contribute to adaptive changes involving cellular cross-talk that enhance proteostasis under low translation conditions. We use the C. elegans system to show that lowering translation in neurons or the germline increases heat shock gene expression and survival under conditions of heat stress. In addition, we find that low translation in these tissues protects motility in a body muscle-specific model of proteotoxicity that results in paralysis. Low translation in neurons or germline also results in increased expression of certain muscle regulatory and structural genes, reversing reduced expression normally observed with aging in C. elegans . Enhanced resilience to protein unfolding stress requires neuronal expression of cbp-1 . Low translation in either neurons or the germline orchestrate protective adaptation in other tissues, including body muscle.

Pediatric cancer is usually fatal1. Once diagnosed, it requires, like any other disease, access to appropriate treatment. Unfortunately, the healthcare system in the Democratic Republic of Congo (DR Congo) is characterized by a lack of specialized staff and health infrastructure; according to the World Health Organization (WHO), Sub-Saharan African countries have just two doctors, 11 nurses or midwives and 10 hospital beds per 10 000 habitants, compared with 32 doctors, 79 nurses or midwives and 63 hospital beds per 10 000 habitants in Europe2. The major challenge in treating cancer in pediatrics remains financial in the DR Congo and in countries with similar conditions. Many low- and middle-income countries rely heavily on out-of-pocket spending to finance healthcare services. This has a negative impact on the use of medical services, with the risk of financial disaster and poverty3. Given this fact, the importance of traditional medicine leads to delays in diagnosis, and the costs are likely to be increased by the complications that are often present at the time of diagnosis, which can also alter the prognosis, contributing to an increase in the mortality rate. The economic and social challenge of treating pediatric cancer, like other chronic diseases such as diabetes and hypertension, in the DR Congo and similar countries, is exacerbated by the fact that, as children are unable to produce economically, it is often their guardians who have to provide primary care4, sometimes forcing them to stop working or reduce their working hours5. The technical challenge remains the absence of appropriate care guidelines (absence of care protocols due to the lack of the necessary technical resources and a national health policy defined on the issue); the low level of education, the low economic level, and the socio-cultural constraints leading to the trivialization and/or mystification of the disease, which may constitute an obstacle to the implementation and monitoring of diagnostic and therapeutic strategies. Because DR Congo is an ethnoculturally diverse country, life is regulated between the visible and invisible worlds6, which is why the majority of cancer patients resort to traditional medicine, delaying diagnosis and treatment, even when this is sometimes possible. The future of pediatric cancer treatment in DR Congo should focus on research/action, which at least remains hampered by financial difficulties. Hence, there is a need to initiate a program of regular funding for cancer treatment in pediatrics. Other aspects that have not yet been taken into consideration will need to be integrated into the management of the disease, in particular holistic management that also includes the family and psychological support for the latter, and palliative care in some cases, which makes pediatric cancer management a multidisciplinary task. Political decision-makers should focus their attention on redefining the healthcare system, devoting sufficient financial resources to the overall management of cancer by creating specialized structures, equipping existing ones, and educating qualified staff. Finally, there is a need for regular cancer surveillance to identify areas where progress is being made and where more needs to be done to improve cancer treatment in pediatrics. Ethical approval Not applicable. Consent Not applicable. Sources of funding This research did not receive any specific grant from funding commercial or not-for-profit sectors. Author contribution B.B.M.E. and B.G.: conception; A.M.D.: administrative support; C.B.T., B.B.M.E., and B.G.: literature search; B.B.M.E.: manuscript preparation; B.G.: manuscript editing; A.M.D.: supervision. All authors were involved in the manuscript review and the final approval of the manuscript. Authorship All authors attest that they meet the current ICMJE guidelines for authorship. Conflicts of interest disclosure The authors declare no conflicts of interest. Research registration unique identifying number (UIN) Not applicable. Guarantor Bugashane Bwa Mihigo Elie is the guarantor of the work. Provenance and peer review Not commissioned, externally peer-reviewed. Data availability statement Not applicable.

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3’ UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.

ABSTRACTDeficiencies in the human dystrophin glycoprotein complex (DGC), which links the extracellular matrix with the intracellular cytoskeleton, cause muscular dystrophies, a group of incurable disorders associated with heterogeneous muscle, brain and eye abnormalities. Stresses such as nutrient deprivation and aging cause muscle wasting, which can be exacerbated by reduced levels of the DGC in membranes, the integrity of which is vital for muscle health and function. Moreover, the DGC operates in multiple signaling pathways, demonstrating an important function in gene expression regulation. To advance disease diagnostics and treatment strategies, we strive to understand the genetic pathways that are perturbed by DGC mutations. Here, we utilized a Drosophila model to investigate the transcriptomic changes in mutants of four DGC components under temperature and metabolic stress. We identified DGC-dependent genes, stress-dependent genes and genes dependent on the DGC for a proper stress response, confirming a novel function of the DGC in stress-response signaling. This perspective yields new insights into the etiology of muscular dystrophy symptoms, possible treatment directions and a better understanding of DGC signaling and regulation under normal and stress conditions.

The second TransatlanticEarly Career Investigator (ECI) G Protein-CoupledReceptor (GPCR) Symposium was an online scientific meeting gearedat young GPCR investigators, with the primary goal of expanding opportunitiesfor sharing research and networking among trainees in North Americaand Europe. Here, we discuss the format of our meeting, its impact,and the challenges and opportunities facing meetings like it in thefuture.