Distribution and persistence of atypical porcine pestivirus in experimentally inoculated pigs

Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc

2021 Jun 02

Buckley, AC;Falkenberg, SM;Palmer, MV;Arruda, PH;Magstadt, DR;Schwartz, KJ;Gatto, IR;Neill, JD;Arruda, BL;
PMID: 34078182 | DOI: 10.1177/10406387211022683

Atypical porcine pestivirus (APPV) is a cause of congenital tremors (CTs) in piglets and has been found in swine populations around the globe. Although systemic distribution of the virus has been reported, there is limited information regarding viral localization at the cellular level and distribution at the tissue level. We collected multiple tissues from 2-d-old piglets (n = 36) born to sows inoculated at 45 or 62 d of gestation with APPV via 3 simultaneous routes: intravenous, intranasal, and directly in amniotic vesicles. In addition, 2 boars from APPV-inoculated sows with CT were raised and euthanized when 11 mo old. In situ hybridization performed on tissue samples from piglets demonstrated a broad and systemic distribution of viral RNA including endothelial cells, fibroblasts, and smooth muscle. Labeling in tissues was more pronounced in piglet tissues compared to boars, with the notable exception of diffuse labeling of the cerebellum in boars. Presence of APPV in boar tissues well after resolution of clinical signs suggests persistence of APPV similar to other pestiviruses.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus

The Journal of comparative neurology

2021 May 19

Levy, J;Droz-Bartholet, F;Achour, M;Facchinetti, P;Parratte, B;Giuliano, F;
PMID: 34008871 | DOI: 10.1002/cne.25191

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Long Noncoding RNA NEAT1: A Potential Biomarker in the Progression of Laryngeal Squamous Cell Carcinoma

ORL; journal for oto-rhino-laryngology and its related specialties

2021 Apr 08

Wang, P;Li, QY;Sun, YN;Wang, JT;Liu, M;
PMID: 33831864 | DOI: 10.1159/000515228

Laryngeal squamous cell carcinoma (LSCC) is diverse in its natural history and responsiveness to treatments. There is an urgent need to generate candidate biomarkers for the stratification and individualization of treatment to avoid overtreatment or inadequate treatment. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been identified as an oncogenic gene in multiple human tumors entitles, and dysregulation of NEAT1 was tightly linked to carcinogenesis and cancer progression. One hundred two paraffin samples of LSCC patients were collected. Furthermore, in situ hybridization (ISH), Kaplan-Meier, and MTT were used to analyze the relationship between NEAT1 and the progress of LSCC. In this study, ISH revealed that NEAT1 was strongly expressed in the nucleus. The increased expression of NEAT1 was correlated with T grade, neck nodal metastasis, clinical stage, drinking history, or smoking history of LSCC. The Kaplan-Meier analysis indicated that patients with higher NEAT1 expression had a worse overall survival in LSCC patients. In addition, NEAT1 knockdown significantly inhibited the growth of LSCC cells. Together, these results suggested that NEAT1 involved in the progress of LSCC and might act as a tumor oncogenic gene. This study provides a potential new marker and target for gene therapy in the treatment of LSCC.

Defects in KCNJ16 Cause a Novel Tubulopathy with Hypokalemia, Salt Wasting, Disturbed Acid-Base Homeostasis, and Sensorineural Deafness

Journal of the American Society of Nephrology : JASN

2021 Apr 02

Schlingmann, KP;Renigunta, A;Hoorn, EJ;Forst, AL;Renigunta, V;Atanasov, V;Mahendran, S;Barakat, TS;Gillion, V;Godefroid, N;Brooks, AS;Lugtenberg, D;Lake, J;Debaix, H;Rudin, C;Knebelmann, B;Tellier, S;Rousset-Rouvière, C;Viering, D;deBaaij, JHF;Weber, S;Palygin, O;Staruschenko, A;Kleta, R;Houillier, P;Bockenhauer, D;Devuyst, O;Vargas-Poussou, R;Warth, R;Zdebik, AA;Konrad, M;
PMID: 33811157 | DOI: 10.1681/ASN.2020111587

The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na+,K+-ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness. A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants. We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents. Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption.

NLRP3 inflammasome induces CD4+ T cell loss in chronically HIV-1-infected patients

The Journal of clinical investigation

2021 Mar 15

Zhang, C;Song, JW;Huang, HH;Fan, X;Huang, L;Deng, JN;Tu, B;Wang, K;Li, J;Zhou, MJ;Yang, CX;Zhao, QW;Yang, T;Wang, LF;Zhang, JY;Xu, RN;Jiao, YM;Shi, M;Shao, F;Sékaly, RP;Wang, FS;
PMID: 33720048 | DOI: 10.1172/JCI138861

Chronic HIV-1 infection is generally characterized by progressive CD4+ T cell depletion due to direct and bystander death that is closely associated with persistent HIV-1 replication and an inflammatory environment in vivo. The mechanisms underlying the loss of CD4+ T cells in patients with chronic HIV-1 infection are incompletely understood. In this study, we simultaneously monitored caspase-1 and caspase-3 activation in circulating CD4+ T cells, which revealed that pyroptotic and apoptotic CD4+ T cells are distinct cell populations with different phenotypic characteristics. Levels of pyroptosis and apoptosis in CD4+ T cells were significantly elevated during chronic HIV-1 infection, and decreased following effective antiretroviral therapy. Notably, the occurrence of pyroptosis was further confirmed by elevated gasdermin D activation in lymph nodes of HIV-1-infected individuals. Mechanistically, caspase-1 activation closely correlated with the inflammatory marker expression and was shown to occur through NLRP3 inflammasome activation driven by virus-dependent and/or -independent ROS production, while caspase-3 activation in CD4+ T cells was more closely related to T cell activation status. Hence, our findings show that NLRP3-dependent pyroptosis plays an essential role in CD4+ T cell loss in HIV-1-infected patients and implicate pyroptosis signaling as a target for anti-HIV-1 treatment.

Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors

Neuron

2021 Mar 11

Yu, W;Pati, D;Pina, MM;Schmidt, KT;Boyt, KM;Hunker, AC;Zweifel, LS;McElligott, ZA;Kash, TL;
PMID: 33740416 | DOI: 10.1016/j.neuron.2021.03.001

Sex differences in pain severity, response, and pathological susceptibility are widely reported, but the neural mechanisms that contribute to these outcomes remain poorly understood. Here we show that dopamine (DA) neurons in the ventrolateral periaqueductal gray/dorsal raphe (vlPAG/DR) differentially regulate pain-related behaviors in male and female mice through projections to the bed nucleus of the stria terminalis (BNST). We find that activation of vlPAG/DRDA+ neurons or vlPAG/DRDA+ terminals in the BNST reduces nociceptive sensitivity during naive and inflammatory pain states in male mice, whereas activation of this pathway in female mice leads to increased locomotion in the presence of salient stimuli. We additionally use slice physiology and genetic editing approaches to demonstrate that vlPAG/DRDA+ projections to the BNST drive sex-specific responses to pain through DA signaling, providing evidence of a novel ascending circuit for pain relief in males and contextual locomotor response in females.

Slc1a3-2A-CreERT2 mice reveal unique features of Bergmann glia and augment a growing collection of Cre drivers and effectors in the 129S4 genetic background

Scientific reports

2021 Mar 08

Kaczmarczyk, L;Reichenbach, N;Blank, N;Jonson, M;Dittrich, L;Petzold, GC;Jackson, WS;
PMID: 33686166 | DOI: 10.1038/s41598-021-84887-2

Genetic variation is a primary determinant of phenotypic diversity. In laboratory mice, genetic variation can be a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains must be made with caution, especially when working with complex phenotypes and disease models. Here we compared behavioral characteristics of C57Bl/6-the strain most widely used in biomedical research-with those of 129S4. In contrast to 129S4, C57Bl/6 demonstrated high within-strain and intra-litter behavioral hyperactivity. Although high consistency would be advantageous, the majority of disease models and transgenic tools are in C57Bl/6. We recently established six Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre line to study astrocytes in 129S4. It was validated with two Cre effector lines: calcium indicator gCaMP5g-tdTomato and RiboTag-a tool widely used to study cell type-specific translatomes. These reporters are in different genomic loci, and in both the Cre was functional and astrocyte-specific. We found that calcium signals lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes, genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.

Genetic disruption of zebrafish mab21l1 reveals a conserved role in eye development and affected pathways

Developmental dynamics : an official publication of the American Association of Anatomists

2021 Feb 11

Seese, SE;Deml, B;Muheisen, S;Sorokina, E;Semina, EV;
PMID: 33570754 | DOI: 10.1002/dvdy.312

The male-abnormal 21 like (MAB21L) genes are important in human ocular development. Homozygous loss of MAB21L1 leads to corneal dystrophy in all affected individuals along with cataracts and buphthalmos in some. The molecular function and downstream pathways of MAB21L factors are largely undefined. We generated the first mab21l1 zebrafish mutant carrying a putative loss-of-function allele, c.107delA p.(Lys36Argfs*7). At the final stages of embryonic development, homozygous mab21l1c.107delA fish displayed enlarged anterior chambers and corneal thinning which progressed with age. Additional studies revealed increased cell death in the mutant corneas, transformation of the cornea into a skin-like epithelium, and progressive lens degeneration with development of fibrous masses in the anterior chamber. RNA-seq of wild-type and mutant ocular transcriptomes revealed significant changes in expression of several genes, including irf1a and b, stat1, elf3, krt17, tlr9 and loxa associated with immunity and/or corneal function. Abnormal expression of lysyl oxidases have been previously linked with corneal thinning, fibrosis, and lens defects in mammals, suggesting a role for loxa misexpression in the progressive mab21l1c.107delA eye phenotype. Zebrafish mab21l1 is essential for normal corneal development, similar to human MAB21L1. The identified molecular changes in mab21l1c.107delA mutants provide the first clues about possible affected pathways. This article is protected by

Local Synthesis of Sarcolemma and Sarcoplasmic Reticulum Membrane Proteins in Cardiac Myocytes

Biophysical Journal

2021 Feb 01

Bogdanov, V;Soltisz, a;Ivanova, M;Andreev, I;Sakuta, G;Davis, J;Veeraraghavan, R;Gyorke, S;
| DOI: 10.1016/j.bpj.2020.11.557

Classically, cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) membrane proteins are thought to be synthesized and processed on the rough ER, in the perinuclear region and then transported to locations of employment in the SR and SL. However, this view is largely based on studies in nonmyocyte cell types. Therefore, we investigated the localization and regulation of synthesis of several key sarcolemmal SL and SR membrane proteins in adult cardiac myocytes. in contrast to the canonical view, in mouse ventricular myocytes we identified synthesis machinery and translation for such proteins, namely SERCA2a, RyR2, Cav1.2 and Nav1.5 localized not only at the perinuclear area, but spread throughout the entire cell volume, particularly in the vicinity of surface/T-tubule and SR membranes. inhibition of microtubular trafficking by colchicine resulted in redistribution of the mRNA signal for these proteins towards the nuclei, consistent with microtubule trafficking providing a dedicated pool of mRNA for local protein translation and synthesis. Active translation of SERCA2a mRNA throughout the cardiac myocyte space was confirmed through visualization of active translation sites using a novel insitu hybridization assay, which we developed. Collectively, these results suggest that synthesis of SR and SL proteins in cardiomyocytes occurs locally at sites of utilization from dedicated, local mRNA pools.

Tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding neuropeptides via Lgr3/8 in the brain

Nature cell biology

2021 Feb 01

Yeom, E;Shin, H;Yoo, W;Jun, E;Kim, S;Hong, SH;Kwon, DW;Ryu, TH;Suh, JM;Kim, SC;Lee, KS;Yu, K;
PMID: 33558728 | DOI: 10.1038/s41556-020-00628-z

In patients with advanced-stage cancer, cancer-associated anorexia affects treatment success and patient survival. However, the underlying mechanism is poorly understood. Here, we show that Dilp8, a Drosophila homologue of mammalian insulin-like 3 peptide (INSL3), is secreted from tumour tissues and induces anorexia through the Lgr3 receptor in the brain. Activated Dilp8-Lgr3 signalling upregulated anorexigenic nucleobinding 1 (NUCB1) and downregulated orexigenic short neuropeptide F (sNPF) and NPF expression in the brain. In the cancer condition, the protein expression of Lgr3 and NUCB1 was significantly upregulated in neurons expressing sNPF and NPF. INSL3 levels were increased in tumour-implanted mice and INSL3-treated mouse hypothalamic cells showed Nucb2 upregulation and Npy downregulation. Food consumption was significantly reduced in intracerebrospinal INSL3-injected mice. In patients with pancreatic cancer, higher serum INSL3 levels increased anorexia. These results indicate that tumour-derived Dilp8/INSL3 induces cancer anorexia by regulating feeding hormones through the Lgr3/Lgr8 receptor in Drosophila and mammals.

Evidence of SARS-CoV-2 Replication and Tropism in the Lungs, Airways and Vascular Endothelium of Patients with Fatal COVID-19: An Autopsy Case-Series

The Journal of infectious diseases

2021 Jan 27

Bhatnagar, J;Gary, J;Reagan-Steiner, S;Estetter, LB;Tong, S;Tao, Y;Denison, AM;Lee, E;DeLeon-Carnes, M;Li, Y;Uehara, A;Paden, CR;Leitgeb, B;Uyeki, TM;Martines, RB;Ritter, JM;Paddock, CD;Shieh, WJ;Zaki, SR;
PMID: 33502471 | DOI: 10.1093/infdis/jiab039

The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of SARS-CoV-2 tropism and replication in various tissues. We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case-patients (age range: 1 month to 84 years; COVID-19 confirmed n=21, suspected n=43) by SARS-CoV-2 RT-PCR. For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in-situ hybridization (ISH), subgenomic RNA RT-PCR, and whole genome sequencing. SARS-CoV-2 was identified by RT-PCR in 32 case-patients (confirmed n=21 and suspected n=11). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR-positive case-patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes and macrophages of lungs, epithelial cells of airways, and in endothelial cells and vessels wall of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. D614G variant was detected in 9 RT-PCR-positive case-patients. We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes.

Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton

Development (Cambridge, England)

2021 Jan 25

Smeeton, J;Natarajan, N;Naveen Kumar, A;Miyashita, T;Baddam, P;Fabian, P;Graf, D;Crump, JG;
PMID: 33462117 | DOI: 10.1242/dev.193409

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

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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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