S3.3c Alveolar macrophage-mediated host resistance against Aspergillus fumigatus

Medical Mycology

2022 Sep 20

Obar, J;
| DOI: 10.1093/mmy/myac072.s3.3c

S3.3 Innate immune responses to pathogenic fungi, September 21, 2022, 4:45 PM - 6:15 PM Alveolar macrophages (AlvMφ) reside on the luminal surface of the airways serving as the primary phagocyte within the airways of the lungs where they act as immune sentinel cells sensing and responding to microbial and environmental exposures. In this role, AlvMφ must be able to respond in a manner that is appropriate to the threat posed which has been hypothesized to occur through sensing microbial vitality and/or patterns of pathogenesis. It is well-established that AlvMφ interact with phagocytose and respond to A. fumigatus, but their role in host resistance against A. fumigatus is currently controversial. Here I will discuss the role of AlvMφ play in orchestrating a robust and effective antifungal innate immune response to mediate A. fumigatus clearance. AlvMφ orchestrate the protective innate immune response against A. fumigatus by sensing live fungal conidia using the cytosolic RNA-sensing MDA5 receptor to initiate the host protective type I and type III interferon response in both mice and humans. The activation of MDA5/MAVS signaling appears to be mediated by both fungal dsRNA-dependent and fungal dsRNA-independent mechanisms. Thus, AlvMφ serve as a central hub for regulating and tuning the antifungal immune response within the respiratory tract.

Consensus guidelines on the construct validity of rodent models of restless legs syndrome

Disease models & mechanisms

2022 Aug 01

Salminen, AV;Clemens, S;García-Borreguero, D;Ghorayeb, I;Li, Y;Manconi, M;Ondo, W;Rye, D;Siegel, JM;Silvani, A;Winkelman, JW;Allen, RP;Ferré, S;International Restless Legs Syndrome Study Group (IRLSSG), ;
PMID: 35946581 | DOI: 10.1242/dmm.049615

Our understanding of the causes and natural course of restless legs syndrome (RLS) is incomplete. The lack of objective diagnostic biomarkers remains a challenge for clinical research and for the development of valid animal models. As a task force of preclinical and clinical scientists, we have previously defined face validity parameters for rodent models of RLS. In this article, we establish new guidelines for the construct validity of RLS rodent models. To do so, we first determined and agreed on the risk, and triggering factors and pathophysiological mechanisms that influence RLS expressivity. We then selected 20 items considered to have sufficient support in the literature, which we grouped by sex and genetic factors, iron-related mechanisms, electrophysiological mechanisms, dopaminergic mechanisms, exposure to medications active in the central nervous system, and others. These factors and biological mechanisms were then translated into rodent bioequivalents deemed to be most appropriate for a rodent model of RLS. We also identified parameters by which to assess and quantify these bioequivalents. Investigating these factors, both individually and in combination, will help to identify their specific roles in the expression of rodent RLS-like phenotypes, which should provide significant translational implications for the diagnosis and treatment of RLS.

Microglia-derived PDGFB promotes neuronal potassium currents to suppress basal sympathetic tonicity and limit hypertension

Immunity

2022 Jul 12

Bi, Q;Wang, C;Cheng, G;Chen, N;Wei, B;Liu, X;Li, L;Lu, C;He, J;Weng, Y;Yin, C;Lin, Y;Wan, S;Zhao, L;Xu, J;Wang, Y;Gu, Y;Shen, XZ;Shi, P;
PMID: 35863346 | DOI: 10.1016/j.immuni.2022.06.018

Although many studies have addressed the regulatory circuits affecting neuronal activities, local non-synaptic mechanisms that determine neuronal excitability remain unclear. Here, we found that microglia prevented overactivation of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) at steady state. Microglia constitutively released platelet-derived growth factor (PDGF) B, which signaled via PDGFRα on neuronal cells and promoted their expression of Kv4.3, a key subunit that conducts potassium currents. Ablation of microglia, conditional deletion of microglial PDGFB, or suppression of neuronal PDGFRα expression in the PVN elevated the excitability of pre-sympathetic neurons and sympathetic outflow, resulting in a profound autonomic dysfunction. Disruption of the PDGFBMG-Kv4.3Neuron pathway predisposed mice to develop hypertension, whereas central supplementation of exogenous PDGFB suppressed pressor response when mice were under hypertensive insult. Our results point to a non-immune action of resident microglia in maintaining the balance of sympathetic outflow, which is important in preventing cardiovascular diseases.

miR-181b targets semaphorin 3A to mediate TGF-β-induced endothelial-mesenchymal transition related to atrial fibrillation

The Journal of clinical investigation

2022 Jul 01

Lai, YJ;Tsai, FC;Chang, GJ;Chang, SH;Huang, CC;Chen, WJ;Yeh, YH;
PMID: 35775491 | DOI: 10.1172/JCI142548

Atrial fibrosis is an essential contributor to atrial fibrillation (AF). It remains unclear whether atrial endocardial endothelial cells (AEECs) that undergo endothelial-mesenchymal transition (EndMT) are among the sources of atrial fibroblasts. We studied human atria, TGF-β-treated human AEECs, cardiac-specific TGF-β-transgenic mice, and heart failure rabbits to identify the underlying mechanism of EndMT in atrial fibrosis. Using isolated AEECs, we found that miR-181b was induced in TGF-β-treated AEECs, which decreased semaphorin 3A (Sema3A) and increased EndMT markers, and these effects could be reversed by a miR-181b antagomir. Experiments in which Sema3A was increased by a peptide or decreased by a siRNA in AEECs revealed a mechanistic link between Sema3A and LIM-kinase 1/phosphorylated cofilin (LIMK/p-cofilin) signaling and suggested that Sema3A is upstream of LIMK in regulating actin remodeling through p-cofilin. Administration of the miR-181b antagomir or recombinant Sema3A to TGF-β-transgenic mice evoked increased Sema3A, reduced EndMT markers, and significantly decreased atrial fibrosis and AF vulnerability. Our study provides a mechanistic link between the induction of EndMT by TGF-β via miR-181b/Sema3A/LIMK/p-cofilin signaling to atrial fibrosis. Blocking miR-181b and increasing Sema3A are potential strategies for AF therapeutic intervention.

VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle

Neuron

2022 Jul 21

Yin, L;Hashikawa, K;Hashikawa, Y;Osakada, T;Lischinsky, JE;Diaz, V;Lin, D;
PMID: 35896109 | DOI: 10.1016/j.neuron.2022.06.026

Sexual behavior is fundamental for the survival of mammalian species and thus supported by dedicated neural substrates. The ventrolateral part of ventromedial hypothalamus (VMHvl) is an essential locus for controlling female sexual behaviors, but recent studies revealed the molecular complexity and functional heterogeneity of VMHvl cells. Here, we identify the cholecystokinin A receptor (Cckar)-expressing cells in the lateral VMHvl (VMHvllCckar) as the key controllers of female sexual behaviors. The inactivation of VMHvllCckar cells in female mice diminishes their interest in males and sexual receptivity, whereas activating these cells has the opposite effects. Female sexual behaviors vary drastically over the reproductive cycle. In vivo recordings reveal reproductive-state-dependent changes in VMHvllCckar cell spontaneous activity and responsivity, with the highest activity occurring during estrus. These in vivo response changes coincide with robust alternation in VMHvllCckar cell excitability and synaptic inputs. Altogether, VMHvllCckar cells represent a key neural population dynamically controlling female sexual behaviors over the reproductive cycle.

Prss29 Cre recombinase mice are useful to study adult uterine gland function

Genesis (New York, N.Y. : 2000)

2022 Jul 22

Kelleher, AM;Allen, CC;Davis, DJ;Spencer, TE;
PMID: 35866844 | DOI: 10.1002/dvg.23493

All mammalian uteri contain glands in their endometrium that develop only or primarily after birth. In mice, those endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful for the study of uterine gland function during pregnancy. Utilizing CRISPR-Cas9 genome editing, Cre recombinase was inserted into the endogenous serine protease 29 precursor (Prss29) gene. Both Prss29 mRNA and Cre recombinase activity was specific to the GE of the mouse uterus following implantation, but was absent from other areas of the female reproductive tract. Next, Prss29-Cre mice were crossed with floxed forkhead box A2 (Foxa2) mice to conditionally delete Foxa2 specifically in the endometrial glands. Foxa2 was absent in the glands of the post-implantation uterus, and Foxa2 deleted mice exhibited complete infertility after their first pregnancy. These results establish that Prss29-Cre mice are a valuable resource to elucidate and explore the functions of glands in the adult uterus.

Data Processing and Germline Variant Calling with the Sentieon Pipeline

Methods in molecular biology (Clifton, N.J.)

2022 Jun 25

Aldana, R;Freed, D;
PMID: 35751805 | DOI: 10.1007/978-1-0716-2293-3_1

Public and private genomic sequencing initiatives generate ever-increasing amounts of genomic data creating a need for improved solutions for genomics data processing (Stephens et al.PLoS Biol 13:e1002195, 2015). The Sentieon Genomics software enables rapid and accurate analysis of next-generation sequence data. In this work, we present a typical use of the Sentieon Genomics software for germline variant calling. The Sentieon germline variant calling pipeline produces more accurate results than other tools on third-party benchmarks (Katherine et al. Front Genet 10:736, 2019; Shen et al. bioRxiv, 885517, 2019) in one tenth the time of comparable pipelines. Parts of this guide come from the official Sentieon Genomics software manual in https://support.sentieon.com/manual (Sentieon. Sentieon Genomics software manual, n.d.) and from the official Sentieon Genomics software application notes in https://support.sentieon.com/appnotes (Sentieon. Sentieon Genomics software application notes, n.d.) and are republished with permission. For additional details and advanced usage instructions of the Sentieon tools, refer to the software manual.

Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination

Nature communications

2022 May 04

Ghorbani, S;Jelinek, E;Jain, R;Buehner, B;Li, C;Lozinski, BM;Sarkar, S;Kaushik, DK;Dong, Y;Wight, TN;Karimi-Abdolrezaee, S;Schenk, GJ;Strijbis, EM;Geurts, J;Zhang, P;Ling, CC;Yong, VW;
PMID: 35508608 | DOI: 10.1038/s41467-022-30032-0

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

Long-Term Precision Editing of Neural Circuits Using Engineered Gap Junction Hemichannels

Biological Psychiatry

2022 May 01

Dzirasa, K;Ransey, E;Chesnov, K;Wisdom, E;Bowman, R;Rodriguez, T;Adamson, E;Thomas, G;Almoril-Porras, A;Schwennesen, H;Colón-Ramos, D;Hultman, R;Bursac, N;
| DOI: 10.1016/j.biopsych.2022.02.055

Background The coordination of activity between brain cells is a key determinant of neural circuit function; nevertheless, approaches that selectively regulate communication between two distinct cellular components of a circuit, while leaving the activity of the presynaptic brain cell undisturbed remain sparse. Methods To address this gap, we developed a novel class of electrical synapses by selectively engineering two connexin proteins found in Morone americana (white perch fish): connexin34.7 (Cx34.7) and connexin35 (Cx35). Results By iteratively exploiting protein mutagenesis, a novel in vitro assay of connexin docking, and computational modeling of connexin hemichannel interactions, we uncovered the pattern of structural motifs that broadly determine connexin hemichannel docking. We then utilized this knowledge to design Cx34.7 and Cx35 hemichannels that dock with each other, but not with themselves nor other major connexins expressed in the human central nervous system. We validated these hemichannels in vivo, demonstrating that they facilitate communication between two neurons in Caenorhabditis elegans and recode a learned behavioral preference. Conclusions This system can be applied to edit circuits composed by pairs of genetically defined brain cell types across multiple species. Thus, we establish a potentially translational approach, ‘Long-term integration of Circuits using connexins’ (LinCx), for context-precise circuit-editing with unprecedented spatiotemporal specificity.

Fat3 Acts Through Independent Cytoskeletal Effectors to Coordinate Asymmetric Cell Behaviors During Polarized Circuit Assembly

SSRN Electronic Journal

2021 Sep 08

Aviles, E;Krol, A;Henle, S;Burroughs-Garcia, J;Deans, M;Goodrich, L;
| DOI: 10.2139/ssrn.3917159

The polarized flow of information through neural circuits depends on the orderly arrangement of neurons, their processes, and their synapses. This polarity emerges sequentially in development, starting with directed migration of neuronal precursors, which subsequently elaborate neurites that form synapses in specific locations. In other organs, Fat cadherins sense position and then polarize individual cells by inducing localized changes in the cytoskeleton that are coordinated across the tissue. Here, we show that the Fat-related protein Fat3 plays an analogous role during assembly of polarized circuits in the murine retina. We found that the Fat3 intracellular domain binds to cytoskeletal regulators and synaptic proteins, with discrete motifs required for amacrine cell migration and neurite retraction. Moreover, upon ICD deletion, extra neurites formed but did not make extra synapses, suggesting that Fat3 independently regulates synapse localization. Thus, Fat3 serves as a molecular node to coordinate asymmetric cell behaviors across development.

The Diminishment of Novel Endometrial Carcinoma-Derived Stem-like Cells by Targeting Mitochondrial Bioenergetics and MYC

International journal of molecular sciences

2022 Feb 22

Helweg, LP;Windmöller, BA;Burghardt, L;Storm, J;Förster, C;Wethkamp, N;Wilkens, L;Kaltschmidt, B;Banz-Jansen, C;Kaltschmidt, C;
PMID: 35269569 | DOI: 10.3390/ijms23052426

Cancer stem cells (CSCs) are a small subpopulation of tumor cells harboring properties that include self-renewal, multi-lineage differentiation, tumor reconstitution, drug resistance and invasiveness, making them key players in tumor relapse. In the present paper, we develop new CSC models and analyze the molecular pathways involved in survival to identify targets for the establishment of novel therapies. Endometrial carcinoma-derived stem-like cells (ECSCs) were isolated from carcinogenic gynecological tissue and analyzed regarding their expression of prominent CSC markers. Further, they were treated with the MYC-signaling inhibitor KJ-Pyr-9, chemotherapeutic agent carboplatin and type II diabetes medication metformin. ECSC populations express common CSC markers, such as Prominin-1 and CD44 antigen as well as epithelial-to-mesenchymal transition markers, Twist, Snail and Slug, and exhibit the ability to form free-floating spheres. The inhibition of MYC signaling and treatment with carboplatin as well as metformin significantly reduced the cell survival of ECSC-like cells. Further, treatment with metformin significantly decreased the mitochondrial membrane potential of ECSC-like cells, while the extracellular lactate concentration was increased. The established ECSC-like populations represent promising in vitro models to further study the contribution of ECSCs to endometrial carcinogenesis. Targeting MYC signaling as well as mitochondrial bioenergetics has shown promising results in the diminishment of ECSCs, although molecular signaling pathways need further investigations.

Curated variation benchmarks for challenging medically relevant autosomal genes

Nature biotechnology

2022 Feb 07

Wagner, J;Olson, ND;Harris, L;McDaniel, J;Cheng, H;Fungtammasan, A;Hwang, YC;Gupta, R;Wenger, AM;Rowell, WJ;Khan, ZM;Farek, J;Zhu, Y;Pisupati, A;Mahmoud, M;Xiao, C;Yoo, B;Sahraeian, SME;Miller, DE;Jáspez, D;Lorenzo-Salazar, JM;Muñoz-Barrera, A;Rubio-Rodríguez, LA;Flores, C;Narzisi, G;Evani, US;Clarke, WE;Lee, J;Mason, CE;Lincoln, SE;Miga, KH;Ebbert, MTW;Shumate, A;Li, H;Chin, CS;Zook, JM;Sedlazeck, FJ;
PMID: 35132260 | DOI: 10.1038/s41587-021-01158-1

The repetitive nature and complexity of some medically relevant genes poses a challenge for their accurate analysis in a clinical setting. The Genome in a Bottle Consortium has provided variant benchmark sets, but these exclude nearly 400 medically relevant genes due to their repetitiveness or polymorphic complexity. Here, we characterize 273 of these 395 challenging autosomal genes using a haplotype-resolved whole-genome assembly. This curated benchmark reports over 17,000 single-nucleotide variations, 3,600 insertions and deletions and 200 structural variations each for human genome reference GRCh37 and GRCh38 across HG002. We show that false duplications in either GRCh37 or GRCh38 result in reference-specific, missed variants for short- and long-read technologies in medically relevant genes, including CBS, CRYAA and KCNE1. When masking these false duplications, variant recall can improve from 8% to 100%. Forming benchmarks from a haplotype-resolved whole-genome assembly may become a prototype for future benchmarks covering the whole genome.

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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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