Structural evidence for visual arrestin priming via complexation of phosphoinositols

Structure (London, England : 1993)

2021 Oct 21

Sander, CL;Luu, J;Kim, K;Furkert, D;Jang, K;Reichenwallner, J;Kang, M;Lee, HJ;Eger, BT;Choe, HW;Fiedler, D;Ernst, OP;Kim, YJ;Palczewski, K;Kiser, PD;
PMID: 34678158 | DOI: 10.1016/j.str.2021.10.002

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail.

Microglia and BDNF at the crossroads of stressor related disorders: Towards a unique trophic phenotype

Neuroscience and biobehavioral reviews

2021 Sep 16

Prowse, N;Hayley, S;
PMID: 34537262 | DOI: 10.1016/j.neubiorev.2021.09.018

Stressors ranging from psychogenic/social to neurogenic/injury to systemic/microbial can impact microglial inflammatory processes, but less is known regarding their effects on trophic properties of microglia. Recent studies do suggest that microglia can modulate neuronal plasticity, possibly through brain derived neurotrophic factor (BDNF). This is particularly important given the link between BDNF and neuropsychiatric and neurodegenerative pathology. We posit that certain activated states of microglia play a role in maintaining the delicate balance of BDNF release onto neuronal synapses. This focused review will address how different "activators" influence the expression and release of microglial BDNF and address the question of tropomyosin receptor kinase B (TrkB) expression on microglia. We will then assess sex-based differences in microglial function and BDNF expression, and how microglia are involved in the stress response and related disorders such as depression. Drawing on research from a variety of other disorders, we will highlight challenges and opportunities for modulators that can shift microglia to a "trophic" phenotype with a view to potential therapeutics relevant for stressor-related disorders.

Macrophage-derived interleukin-6 is necessary and sufficient for choroidal angiogenesis

Scientific reports

2021 Sep 10

Droho, S;Cuda, CM;Perlman, H;Lavine, JA;
PMID: 34508129 | DOI: 10.1038/s41598-021-97522-x

Neovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Interleukin-6 (IL6) is a pro-inflammatory and pro-angiogenic cytokine that is correlated with AMD progression and nAMD activity. We hypothesize that anti-IL6 therapy is a potential nAMD therapeutic. We found that IL6 levels were increased after laser injury and expressed by macrophages. Il6-deficiency decreased laser-induced CNV area and exogenous IL6 addition increased choroidal sprouting angiogenesis. Il6-null mice demonstrated equally increased macrophage numbers as wildtype mice. At steady state, IL6R expression was detected on peripheral blood and ocular monocytes. After laser injury, the number of IL6R+Ly6C+ monocytes in blood and IL6R+ macrophages in the eye were increased. In human choroid, macrophages expressed IL6, IL6R, and IL6ST. Furthermore, IL6R+ macrophages displayed a transcriptional profile consistent with STAT3 (signal transducer and activator of transcription 3) activation and angiogenesis. Our data show that IL6 is both necessary and sufficient for choroidal angiogenesis. Macrophage-derived IL6 may stimulate choroidal angiogenesis via classical activation of IL6R+ macrophages, which then stimulate angiogenesis. Targeting IL6 or the IL6R could be an effective adjunctive therapy for treatment-resistant nAMD patients.

Diversity of developing peripheral glia revealed by single-cell RNA sequencing

Developmental cell

2021 Aug 26

Tasdemir-Yilmaz, OE;Druckenbrod, NR;Olukoya, OO;Dong, W;Yung, AR;Bastille, I;Pazyra-Murphy, MF;Sitko, AA;Hale, EB;Vigneau, S;Gimelbrant, AA;Kharchenko, PV;Goodrich, LV;Segal, RA;
PMID: 34469751 | DOI: 10.1016/j.devcel.2021.08.005

The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.

The Use of Paraffin Blocks/Pathology Archives for Clinical Biobanking

Biobanking of Human Biospecimens

2021 Aug 26

Stanta, G;Bonin, S;
| DOI: 10.1007/978-3-030-55901-4_5

Every human tissue collected from surgery or biopsy is formalin-fixed and paraffin-embedded (FFPE). A huge quantity of human tissues is preserved in hospitals, and it is possible to estimate that in Europe about three hundred million new specimens of tissues are stored in archives every year. These archive tissues (AT) represent any type of even rare pathological lesions with a number of cases sufficient for any possible study. Frequent evidence of acquired resistance to the new targeted therapies has posed clinicians new issues to be addressed, such as the need to go back to patients’ tissues. Increasingly more often we have to use ATs because of clinical research, which is now starting to be an integrated process with applied medicine. We cannot separate clinics from clinical research, it is a unique entity. This creates new necessities such as better quality ATs, more standardized methods, a careful choice of the tissues, and a better organization. The pathology archives of hospitals are clinical biorepositories that are different from a research biobank. The major difference is related to the clinical purpose of these archives; even though this may not seem to be an obstacle to their utilization for research purposes, it is perfectly fitted with the most recent necessities of a kind of clinical research that is strictly related to medicine and perfectly integrated into it. The use of AT biorepositories represents a peculiar type of tissue as a source that also needs a different bioethical approach.

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding

Neuron

2021 Aug 11

Han, S;Okawa, S;Wilkinson, GA;Ghazale, H;Adnani, L;Dixit, R;Tavares, L;Faisal, I;Brooks, MJ;Cortay, V;Zinyk, D;Sivitilli, A;Li, S;Malik, F;Ilnytskyy, Y;Angarica, VE;Gao, J;Chinchalongporn, V;Oproescu, AM;Vasan, L;Touahri, Y;David, LA;Raharjo, E;Kim, JW;Wu, W;Rahmani, W;Chan, JA;Kovalchuk, I;Attisano, L;Kurrasch, D;Dehay, C;Swaroop, A;Castro, DS;Biernaskie, J;Del Sol, A;Schuurmans, C;
PMID: 34407390 | DOI: 10.1016/j.neuron.2021.07.007

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.

Spatial omics and multiplexed imaging to explore cancer biology

Nature methods

2021 Aug 02

Lewis, SM;Asselin-Labat, ML;Nguyen, Q;Berthelet, J;Tan, X;Wimmer, VC;Merino, D;Rogers, KL;Naik, SH;
PMID: 34341583 | DOI: 10.1038/s41592-021-01203-6

Understanding intratumoral heterogeneity-the molecular variation among cells within a tumor-promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. Single-cell analyses, especially RNA sequencing and other genomics modalities, have been transformative in revealing novel biomarkers and molecular regulators associated with tumor growth, metastasis and drug resistance. However, these approaches fail to provide a complete picture of tumor biology, as information on cellular location within the tumor microenvironment is lost. New technologies leveraging multiplexed fluorescence, DNA, RNA and isotope labeling enable the detection of tens to thousands of cancer subclones or molecular biomarkers within their native spatial context. The expeditious growth in these techniques, along with methods for multiomics data integration, promises to yield a more comprehensive understanding of cell-to-cell variation within and between individual tumors. Here we provide the current state and future perspectives on the spatial technologies expected to drive the next generation of research and diagnostic and therapeutic strategies for cancer.

Morphological Object Localization: A Novel Image Analysis Pipeline for Quantitative Spatial Localization of Biomolecule Signal from Fluorescence Microscopy Data

Microscopy and Microanalysis

2021 Aug 01

Soltisz, A;Veeraraghavan, R;Bogdanov, V;Gyorke, S;
| DOI: 10.1017/s1431927621009181

The spatial distribution of biomolecules (BMs) within cells and tissues is often a significant determinant of biological / physiological function. Thus, its assessment is not only a ubiquitous feature in the life sciences but also a vital component in the clinical diagnosis and treatment of many pathologies. This typically entails microscopic imaging of fluorescently labeled biomolecules within biological specimens and analysis and interpretation of the resulting images. This is accomplished by methods ranging from qualitative visual assessment of representative images to colocalization analysis, which quantifies the superposition of immunosignals corresponding to co-labeled BMs. These commonly employed approaches have several key limitations. The selection and visual assessment of representative images is subjective and highly susceptible to bias. And, although quantitative, conventional colocalization analysis is susceptible to variations between fluorophores, and oversimplifies complex spatial distributions of BMs into a single numerical index of signal superposition.[1] It provides no information about nonsuperimposed signals, and thus, lacks the sensitivity and selectivity to capture intra- and inter-individual variability or discern subtle forms of biological remodeling, as occurs in the early stages of disease. Here, we present a novel, high-throughput image analysis pipeline, called Morphological Object Localization (MOL), for comprehensive, quantitative spatial localization of BM signals relative to each other as well as structural landmarks from fluorescence microscopy data. This tool offers a quick and user-friendly alternative to current approaches with unprecedented capabilities for quantitative assessment of cell / tissue structure.

Exposure of human fetal kidneys to mild analgesics interferes with early nephrogenesis

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Jul 01

Leverrier-Penna, S;Michel, A;Lecante, LL;Costet, N;Suglia, A;Desdoits-Lethimonier, C;Boulay, H;Viel, R;Chemouny, JM;Becker, E;Lavoué, V;Rolland, AD;Dejucq-Rainsford, N;Vigneau, C;Mazaud-Guittot, S;
PMID: 34105801 | DOI: 10.1096/fj.202100050R

Acetaminophen, aspirin, and ibuprofen are mild analgesics commonly used by pregnant women, the sole current recommendation being to avoid ibuprofen from the fifth month of gestation. The nephrotoxicity of these three analgesics is well documented in adults, as is their interference with prostaglandins biosynthesis. Here we investigated the effect of these analgesics on human first trimester kidneys ex vivo. We first evaluated prostaglandins biosynthesis functionality by performing a wide screening of prostaglandin expression patterns in first trimester human kidneys. We demonstrated that prostaglandins biosynthesis machinery is functional during early nephrogenesis. Human fetal kidney explants aged 7-12 developmental weeks were exposed ex vivo to ibuprofen, aspirin or acetaminophen for 7 days, and analyzed by histology, immunohistochemistry, and flow cytometry. This study has revealed that these analgesics induced a spectrum of abnormalities within early developing structures, ranging from cell death to a decline in differentiating glomeruli density. These results warrant caution for the use of these medicines during the first trimester of pregnancy.

Snail enhances arginine synthesis by inhibiting ubiquitination-mediated degradation of ASS1

EMBO reports

2021 Jun 29

Jia, H;Yang, Y;Li, M;Chu, Y;Song, H;Zhang, J;Zhang, D;Zhang, Q;Xu, Y;Wang, J;Xu, H;Zou, X;Peng, H;Hou, Z;
PMID: 34184805 | DOI: 10.15252/embr.202051780

Snail is a dedicated transcriptional repressor and acts as a master inducer of EMT and metastasis, yet the underlying signaling cascades triggered by Snail still remain elusive. Here, we report that Snail promotes colorectal cancer (CRC) migration by preventing non-coding RNA LOC113230-mediated degradation of argininosuccinate synthase 1 (ASS1). LOC113230 is a novel Snail target gene, and Snail binds to the functional E-boxes within its proximal promoter to repress its expression in response to TGF-β induction. Ectopic expression of LOC113230 potently suppresses CRC cell growth, migration, and lung metastasis in xenograft experiments. Mechanistically, LOC113230 acts as a scaffold to facilitate recruiting LRPPRC and the TRAF2 E3 ubiquitin ligase to ASS1, resulting in enhanced ubiquitination and degradation of ASS1 and decreased arginine synthesis. Moreover, elevated ASS1 expression is essential for CRC growth and migration. Collectively, these findings suggest that TGF-β and Snail promote arginine synthesis via inhibiting LOC113230-mediated LRPPRC/TRAF2/ASS1 complex assembly and this complex can serve as potential target for the development of new therapeutic approaches to treat CRC.

N6-Methyladenosine on mRNA facilitates a phase-separated nuclear body that suppresses myeloid leukemic differentiation

Cancer cell

2021 May 12

Cheng, Y;Xie, W;Pickering, BF;Chu, KL;Savino, AM;Yang, X;Luo, H;Nguyen, DT;Mo, S;Barin, E;Velleca, A;Rohwetter, TM;Patel, DJ;Jaffrey, SR;Kharas, MG;
PMID: 34048709 | DOI: 10.1016/j.ccell.2021.04.017

N6-Methyladenosine (m6A) on mRNAs mediates different biological processes and its dysregulation contributes to tumorigenesis. How m6A dictates its diverse molecular and cellular effects in leukemias remains unknown. We found that YTHDC1 is the essential m6A reader in myeloid leukemia from a genome-wide CRISPR screen and that m6A is required for YTHDC1 to undergo liquid-liquid phase separation and form nuclear YTHDC1-m6A condensates (nYACs). The number of nYACs increases in acute myeloid leukemia (AML) cells compared with normal hematopoietic stem and progenitor cells. AML cells require the nYACs to maintain cell survival and the undifferentiated state that is critical for leukemia maintenance. Furthermore, nYACs enable YTHDC1 to protect m6A-mRNAs from the PAXT complex and exosome-associated RNA degradation. Collectively, m6A is required for the formation of a nuclear body mediated by phase separation that maintains mRNA stability and control cancer cell survival and differentiation.

PRISM: Recovering cell type specific expression profiles from individual composite RNA-seq samples

Bioinformatics (Oxford, England)

2021 Mar 15

Häkkinen, A;Zhang, K;Alkodsi, A;Andersson, N;Pekcan Erkan, E;Dai, J;Kaipio, K;Lamminen, T;Mansuri, N;Huhtinen, K;Vähärautio, A;Carpén, O;Hynninen, J;Hietanen, S;Lehtonen, R;Hautaniemi, S;
PMID: 33720334 | DOI: 10.1093/bioinformatics/btab178

A major challenge in analyzing cancer patient transcriptomes is that the tumors are inherently heterogeneous and evolving. We analyzed 214 bulk RNA samples of a longitudinal, prospective ovarian cancer cohort and found that the sample composition changes systematically due to chemotherapy and between the anatomical sites, preventing direct comparison of treatment-naive and treated samples. To overcome this, we developed PRISM, a latent statistical framework to simultaneously extract the sample composition and cell type specific whole-transcriptome profiles adapted to each individual sample. Our results indicate that the PRISM-derived composition-free transcriptomic profiles and signatures derived from them predict the patient response better than the composite raw bulk data. We validated our findings in independent ovarian cancer and melanoma cohorts, and verified that PRISM accurately estimates the composition and cell type specific expression through whole-genome sequencing and RNA in situ hybridization experiments. https://bitbucket.org/anthakki/prism. Supplementary data are available at Bioinformatics online.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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