Suppressing Sema3A expression in muscle satellite cells affects terminal Schwann cells after muscle and nerve injury

The FASEB Journal

2021 Jan 01

Daneshvar, N;Tatsumi, R;Matsuyoshi, Y;
| DOI: 10.1096/fasebj.2021.35.S1.03277

To examine the role of Sema3A expression in satellite cells (SCs) and terminal Schwann cells (TSCs) in neuromuscular junction (NMJs) formation, expression was investigated during recovery from muscle- or nerve-crush injuries in muscle from mice with a SC-specific knockout of Sema3A. We tested the hypothesis that loss of SC-specific Sema3A expression would disrupt TSC gene and protein expression after both injuries. Gene expression in synaptic areas was studied using RNAscope multiplex fluorescence in situ hybridisation (ISH, Advanced Cell Diagnostics) to examine TSCs (Sema3A, S100B, P75NGFR), Pax7+ SCs, and Westerns to assay proteins (Sema3A and S100B, and γAchR, related to denervation). Muscle from transgenics with tamoxifen-induced conditional knockout, and two control groups (injured non-knockouts and no-surgery SC-specific knockouts) were examined 14 and 21days after nerve crush and 21 and 35days after muscle crush (ethics approval A30-142-0 (Kyushu U) and F14-15 UManitoba). Expression sites (number, area, and intensity) for Sema3A, S100B, P75NGFR and Pax7 mRNA were imaged, measured (Celleste software) and analyzed (ANOVA, linear regression, and Principal Component Analysis, PCA) as a function of regeneration time. After muscle crush, P75NGFR expression was higher in SC-specific Sema3A knockout mice (days 21 and 35) than in non-knockout controls (p

USH2A is a Meissner\'s corpuscle protein necessary for normal vibration sensing in mice and humans

Nature neuroscience

2021 Jan 01

Schwaller, F;Bégay, V;García-García, G;Taberner, FJ;Moshourab, R;McDonald, B;Docter, T;Kühnemund, J;Ojeda-Alonso, J;Paricio-Montesinos, R;Lechner, SG;Poulet, JFA;Millan, JM;Lewin, GR;
PMID: 33288907 | DOI: 10.1038/s41593-020-00751-y

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner's corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner's corpuscles. Pathogenic USH2A mutations cause Usher's syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner's corpuscles, recorded from Ush2a-/- mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.

Keeping it simple: Zebrafish directly sense spinal cord stretch to regulate swimming

Neuron

2021 Apr 07

Calabrese, RL;
PMID: 33831361 | DOI: 10.1016/j.neuron.2021.03.019

Picton et al. show that in zebrafish, a class of neurons located in the spinal cord sense spinal cord stretch during the lateral bends of undulatory swimming and provide inhibition to the swimming motor patterning generating network, thus acting as both mechanosensors and inhibitory interneurons.

Developing Vaccines to Improve Preparedness for Filovirus Outbreaks: The Perspective of the USA Biomedical Advanced Research and Development Authority (BARDA)

Vaccines

2023 Jun 19

Parish, LA;Stavale, EJ;Houchens, CR;Wolfe, DN;
PMID: 37376509 | DOI: 10.3390/vaccines11061120

Outbreaks of viral hemorrhagic fever caused by filoviruses have become more prevalent in recent years, with outbreaks of Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV) all occurring in 2022 and 2023. While licensed vaccines are now available for EBOV, vaccine candidates for SUDV and MARV are all in preclinical or early clinical development phases. During the recent outbreak of SUDV virus disease, the Biomedical Advanced Research and Development Authority (BARDA), as part of the Administration for Strategic Preparedness and Response within the U.S. Department of Health and Human Services, implemented key actions with our existing partners to advance preparedness and enable rapid response to the outbreak, while also aligning with global partners involved in the implementation of clinical trials in an outbreak setting. Beyond pre-existing plans prior to the outbreak, BARDA worked with product sponsors to expedite manufacturing of vaccine doses that could be utilized in clinical trials. While the SUDV outbreak has since ended, a new outbreak of MARV disease has emerged. It remains critical that we continue to advance a portfolio of vaccines against SUDV and MARV while also expediting manufacturing activities ahead of, or in parallel if needed, outbreaks.

Splicing Factor SRSF1 Promotes Pancreatitis and KRASG12D-Mediated Pancreatic Cancer

Cancer discovery

2023 Apr 26

Wan, L;Lin, KT;Rahman, MA;Ishigami, Y;Wang, Z;Jensen, MA;Wilkinson, JE;Park, Y;Tuveson, DA;Krainer, AR;
PMID: 37098965 | DOI: 10.1158/2159-8290.CD-22-1013

Inflammation is strongly associated with pancreatic ductal adenocarcinoma (PDAC), a highly lethal malignancy. Dysregulated RNA splicing factors have been widely reported in tumorigenesis, but their involvement in pancreatitis and PDAC is not well understood. Here, we report that the splicing factor SRSF1 is highly expressed in pancreatitis, PDAC precursor lesions, and tumors. Increased SRSF1 is sufficient to induce pancreatitis and accelerate KRASG12D-mediated PDAC. Mechanistically, SRSF1 activates MAPK signaling-partly by upregulating interleukin 1 receptor type 1 (IL1R1) through alternative-splicing-regulated mRNA stability. Additionally, SRSF1 protein is destabilized through a negative feedback mechanism in phenotypically normal epithelial cells expressing KRASG12D in mouse pancreas, and in pancreas organoids acutely expressing KRASG12D, buffering MAPK signaling and maintaining pancreas-cell homeostasis. This negative-feedback regulation of SRSF1 is overcome by hyperactive MYC, facilitating PDAC tumorigenesis. Our findings implicate SRSF1 in the etiology of pancreatitis and PDAC, and point to SRSF1-misregulated alternative splicing as a potential therapeutic target.

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

Science immunology

2023 Feb 03

Sahu, SK;Ozantürk, AN;Kulkarni, DH;Ma, L;Barve, RA;Dannull, L;Lu, A;Starick, M;McPhatter, J;Garnica, L;Sanfillipo-Burchman, M;Kunen, J;Wu, X;Gelman, AE;Brody, SL;Atkinson, JP;Kulkarni, HS;
PMID: 36735773 | DOI: 10.1126/sciimmunol.abp9547

The complement component C3 is a fundamental plasma protein for host defense, produced largely by the liver. However, recent work has demonstrated the critical importance of tissue-specific C3 expression in cell survival. Here, we analyzed the effects of local versus peripheral sources of C3 expression in a model of acute bacterial pneumonia induced by Pseudomonas aeruginosa. Whereas mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient only in liver-derived C3 remained protected, comparable to wild-type mice. Human lung transcriptome analysis showed that secretory epithelial cells, such as club cells, express high levels of C3 mRNA. Mice with tamoxifen-induced C3 gene ablation from club cells in the lung had worse pulmonary injury compared with similarly treated controls, despite maintaining normal circulating C3 levels. Last, in both the mouse pneumonia model and cultured primary human airway epithelial cells, we showed that stress-induced death associated with C3 deficiency parallels that seen in Factor B deficiency rather than C3a receptor deficiency. Moreover, C3-mediated reduction in epithelial cell death requires alternative pathway component Factor B. Thus, our findings suggest that a pathway reliant on locally derived C3 and Factor B protects the lung mucosal barrier.

Unexpected failure of rod bipolar cell targeting using L7Cre-2 mice

Experimental eye research

2023 Mar 01

Peperstraete, K;Baes, M;Swinkels, D;
PMID: 36740160 | DOI: 10.1016/j.exer.2023.109406

Utilizing cell type-specific knockout mice has been an excellent tool for decades not only to explore the role of a gene in a specific cell, but also to unravel the underlying mechanism in diseases. To investigate the mechanistic association between dysfunction of the peroxisomal protein multifunctional protein 2 (MFP2) and retinopathy, we generated and phenotyped multiple transgenic mouse models with global or cell type-specific MFP2 deletion. These studies pointed to a potential role of MFP2 specifically in rod bipolar cells. To explore this, we aimed to create rod bipolar cell specific knockout mice of Mfp2 by crossing Mfp2L/L mice with L7Cre-2 mice (also known as PCP2Cre), generating L7-Mfp2-/- mice. L7Cre-2 mice express Cre recombinase under the control of the L7 promoter, which is believed to be exclusively expressed in rod bipolar cells and cerebellar Purkinje cells. Unexpectedly, only sporadic Cre activity was observed in the rod bipolar cells of L7-Mfp2-/- mice, despite efficient Cre recombination in cerebellar Purkinje cells. Moreover, a variable fraction of photoreceptors was targeted, which does not correspond with the supposed specificity of L7Cre-2 mice. These observations indicate that L7Cre-2 mice can be exploited to manipulate Purkinje cells in the cerebellum, whereas they cannot be used to generate rod bipolar cell specific knockout mice. For this aim, we suggest utilizing an independently generated mouse line named BAC-L7-IRES-Cre.

Epstein-Barr Virus and the Pathogenesis of Diffuse Large B-Cell Lymphoma

Life (Basel, Switzerland)

2023 Feb 14

Ross, AM;Leahy, CI;Neylon, F;Steigerova, J;Flodr, P;Navratilova, M;Urbankova, H;Vrzalikova, K;Mundo, L;Lazzi, S;Leoncini, L;Pugh, M;Murray, PG;
PMID: 36836878 | DOI: 10.3390/life13020521

Epstein-Barr virus (EBV), defined as a group I carcinogen by the World Health Organization (WHO), is present in the tumour cells of patients with different forms of B-cell lymphoma, including Burkitt lymphoma, Hodgkin lymphoma, post-transplant lymphoproliferative disorders, and, most recently, diffuse large B-cell lymphoma (DLBCL). Understanding how EBV contributes to the development of these different types of B-cell lymphoma has not only provided fundamental insights into the underlying mechanisms of viral oncogenesis, but has also highlighted potential new therapeutic opportunities. In this review, we describe the effects of EBV infection in normal B-cells and we address the germinal centre model of infection and how this can lead to lymphoma in some instances. We then explore the recent reclassification of EBV+ DLBCL as an established entity in the WHO fifth edition and ICC 2022 classifications, emphasising the unique nature of this entity. To that end, we also explore the unique genetic background of this entity and briefly discuss the potential role of the tumour microenvironment in lymphomagenesis and disease progression. Despite the recent progress in elucidating the mechanisms of this malignancy, much work remains to be done to improve patient stratification, treatment strategies, and outcomes.

WT1 regulates expression of DNA-repair gene Neil3 during nephrogenesis

American journal of physiology. Renal physiology

2022 Dec 22

Dickinson, K;Hammond, L;Akpa, M;Chu, LL;Lalonde, CT;Goumba, A;Goodyer, P;
PMID: 36546838 | DOI: 10.1152/ajprenal.00207.2022

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor, WT1, which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA-repair genes that protect the genome. We analyzed transcript levels for a panel of DNA-repair genes in E17.5 vs adult mouse kidneys and noted seven that were increased >20-fold. We then isolated d1(+) NPCs from E17.5 kidneys and found that only one, Neil3, was enriched. RNAscope ISH of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone vs mature nephron structures. To determine whether Neil3-expression is WT1-dependent, we knocked down Wt1 in d1(+) NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 directly binds to the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased two-fold in WT1(+) vs WT1(-) cells. We propose that Neil3 is a WT1-dependent DNA-repair gene, expressed at high levels in d1(+) NPCs where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.

The Immune Cell Profile of the Developing Rat Brain

Brain, behavior, and immunity

2022 Aug 29

Reinl, EL;Blanchard, AC;Graham, EL;Edwards, S;Dionisos, C;McCarthy, MM;
PMID: 36049705 | DOI: 10.1016/j.bbi.2022.08.012

Little is known about the peripheral immune cell (PIC) profile of the developing brain despite growing appreciation for these cells in the mature nervous system. To address this gap, the PIC profile, defined as which cells are present, where they are located, and for how long, was examined in the developing rat using spectral flow cytometry. Select regions of the rat brain (cerebellum, hippocampus, and hypothalamus) were examined at embryonic day 20, and postnatal days 0, 7 and 16. At their peak (E20), PICs were most abundant in the cerebellum, then the hippocampus and hypothalamus. Within the PIC pool, monocytes were most prevalent in all regions and time points, and shifted from being majority classical at E20 to non-classical by PN7. T cells increased over time, and shifted from majority cytotoxic to T-helper cells by PN7. This suggests the PIC profile transitions from reactive to adaptive and surveilling in the second postnatal week. NK cells and mast cells increased temporarily, and mast cells were restricted to the hippocampus and hypothalamus, suggesting they may play a specific role in the development of those regions. Mimicking a viral infection by administration of Poly I:C increased the influx of PICs into the neonatal brain, particularly of NK cells and in the case of males only, non-classical monocytes. This work provides a map for researchers as they study immune cell contributions to healthy and pathological brain development.

Systematic comparison of pancreatic ductal adenocarcinoma models identifies a conserved highly plastic basal cell state

Cancer research

2022 Aug 11

Pitter, KL;Grbovic-Huezo, O;Joost, S;Singhal, A;Blum, M;Wu, K;Holm, M;Ferrena, A;Bhutkar, A;Hudson, A;Lecomte, N;de Stanchina, E;Chaligne, R;Iacobuzio-Donahue, CA;Pe'er, D;Tammela, T;
PMID: 35952360 | DOI: 10.1158/0008-5472.CAN-22-1742

Intra-tumoral heterogeneity and cellular plasticity have emerged as hallmarks of cancer, including pancreatic ductal adenocarcinoma (PDAC). As PDAC portends a dire prognosis, a better understanding of the mechanisms underpinning cellular diversity in PDAC is crucial. Here, we investigated the cellular heterogeneity of PDAC cancer cells across a range of in vitro and in vivo growth conditions using single-cell genomics. Heterogeneity contracted significantly in 2D and 3D cell culture models but was restored upon orthotopic transplantation. Orthotopic transplants reproducibly acquired cell states identified in autochthonous PDAC tumors, including a basal state exhibiting co-expression and co-accessibility of epithelial and mesenchymal genes. Lineage-tracing combined with single-cell transcriptomics revealed that basal cells display high plasticity in situ. This work defines the impact of cellular growth conditions on phenotypic diversity and uncovers a highly plastic cell state with the capacity to facilitate state transitions and promote intra-tumoral heterogeneity in PDAC.

Senecavirus A: Frequently asked questions

Journal of Swine Health and Production

2022 May 02

Buckley, A;Lager, K;
| DOI: 10.54846/jshap/1270

Senecavirus A (SVA) has been demonstrated to be a causative agent for vesicular disease in swine. It is clinically indistinguishable from other agents that cause vesicular disease such as foot-and-mouth disease virus (FMDV), which is a reportable foreign animal disease (FAD). Thus, an investigation is initiated to rule out FMDV every time a vesicle is observed. Senecavirus A has now been reported across the Americas and Asia, and it appears the ecology of this virus has changed from sporadic infections to an endemic disease in some areas. In addition to vesicular disease, there have also been reports of increased neonatal mortality on affected sow farms. Knowledge about the pathogenesis of SVA in swine can provide many benefits to the swine industry. Understanding how long the virus can be detected in various sample types after infection can aide in choosing the correct samples to collect for diagnosis. In addition, the duration of virus shedding can help determine measures to control virus spread between animals. Prevention of SVA infection and disease with an efficacious vaccine could improve swine welfare, minimize SVA transmission, and reduce the burden of FAD investigations.

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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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