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Molecular Analysis of the Kidney From a Patient With COVID-19-Associated Collapsing Glomerulopathy

Kidney medicine

2021 Apr 28

Meliambro, K;Li, X;Salem, F;Yi, Z;Sun, Z;Chan, L;Chung, M;Chancay, J;Vy, HMT;Nadkarni, G;Wong, JS;Fu, J;Lee, K;Zhang, W;He, JC;Campbell, KN;
PMID: 33942030 | DOI: 10.1016/j.xkme.2021.02.012

Recent Case reports suggest COVID-19 is associated with collapsing glomerulopathy in African Americans with APOL1 risk alleles, however, it is unclear if disease pathogenesis is similar to HIVAN. Here RNA sequencing analysis of a kidney biopsy specimen from a patient with COVID-19-associated collapsing glomerulopathy and APOL1 risk alleles (G1/G1) revealed similar levels of APOL1 and ACE2 mRNA transcripts as compared to 12 control kidney samples downloaded from the GTEx Portal. Whole genome sequencing of the COVID-19-associated collapsing glomerulopathy kidney sample identified four indel gene variants, three of which are of unknown significance with respect to chronic kidney disease and/or FSGS. Molecular profiling of the kidney demonstrated activation of COVID-19-associated cell injury pathways such as inflammation and coagulation. Evidence for direct SARS-CoV-2 infection of kidney cells was lacking, which is consistent with the findings of several recent studies. Interestingly, immunostaining of kidney biopsy sections revealed increased expression of phospho-STAT3 in both COVID-19-associated collapsing glomerulopathy and HIVAN as compared to control kidney tissue. Importantly, IL-6-induced activation of STAT3 may be a targetable mechanism driving COVID-19-associated acute kidney injury.

Visualization of intestinal infections with astro- and sapovirus in mink (Neovison vison) kits by in situ hybridization

FEMS Microbes

2021 Apr 28

Birch, J;Leijon, M;Nielsen, S;Struve, T;Jensen, H;
| DOI: 10.1093/femsmc/xtab005

Clarification of the infection microbiology remains a challenge in the pre-weaning diarrhea (PWD) syndrome in farmed mink (Neovison vison). Duodenal, jejunal and colon sections from 36 mink kits with PWD were systematically examined by chromogen in situ hybridization targeting two incriminated viruses: mink astrovirus and mink sapovirus. Using the RNAscope 2.5 HD Duplex Assay, astrovirus and sapovirus were visualized and simultaneously demonstrated in the gut tissue. Both viruses infect enterocytes in the small intestine with a specific localization pattern; astrovirus affects the two apical thirds of the villi, whereas sapovirus generally affects the basal parts of the villi. Furthermore, we demonstrated that astrovirus in mink does not target the goblet cells. This is the first time astro- and calicivirus have been visualized in mink kit gut tissue, and these findings might be important in clarification of the impact of these viruses in the PWD syndrome.

Differentiation reveals latent features of aging and an energy barrier in murine myogenesis

Cell reports

2021 Apr 27

Kimmel, JC;Yi, N;Roy, M;Hendrickson, DG;Kelley, DR;
PMID: 33910007 | DOI: 10.1016/j.celrep.2021.109046

Skeletal muscle experiences a decline in lean mass and regenerative potential with age, in part due to intrinsic changes in progenitor cells. However, it remains unclear how age-related changes in progenitors manifest across a differentiation trajectory. Here, we perform single-cell RNA sequencing (RNA-seq) on muscle mononuclear cells from young and aged mice and profile muscle stem cells (MuSCs) and fibro-adipose progenitors (FAPs) after differentiation. Differentiation increases the magnitude of age-related change in MuSCs and FAPs, but it also masks a subset of age-related changes present in progenitors. Using a dynamical systems approach and RNA velocity, we find that aged MuSCs follow the same differentiation trajectory as young cells but stall in differentiation near a commitment decision. Our results suggest that differentiation reveals latent features of aging and that fate commitment decisions are delayed in aged myogenic cells in vitro.

A cellular and spatial map of the choroid plexus across brain ventricles and ages

Cell

2021 Apr 27

Dani, N;Herbst, RH;McCabe, C;Green, GS;Kaiser, K;Head, JP;Cui, J;Shipley, FB;Jang, A;Dionne, D;Nguyen, L;Rodman, C;Riesenfeld, SJ;Prochazka, J;Prochazkova, M;Sedlacek, R;Zhang, F;Bryja, V;Rozenblatt-Rosen, O;Habib, N;Regev, A;Lehtinen, MK;
PMID: 33932339 | DOI: 10.1016/j.cell.2021.04.003

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1β (IL-1β) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

Enhanced Ca2+ signaling, mild primary aldosteronism, and hypertension in a familial hyperaldosteronism mouse model (Cacna1h\n M1560V/+\n)

Proceedings of the National Academy of Sciences of the United States of America

2021 Apr 27

Seidel, E;Schewe, J;Zhang, J;Dinh, HA;Forslund, SK;Markó, L;Hellmig, N;Peters, J;Muller, DN;Lifton, RP;Nottoli, T;Stölting, G;Scholl, UI;
PMID: 33879608 | DOI: 10.1073/pnas.2014876118

Gain-of-function mutations in the CACNA1H gene (encoding the T-type calcium channel CaV3.2) cause autosomal-dominant familial hyperaldosteronism type IV (FH-IV) and early-onset hypertension in humans. We used CRISPR/Cas9 to generate Cacna1h M1560V/+ knockin mice as a model of the most common FH-IV mutation, along with corresponding knockout mice (Cacna1h -/- ). Adrenal morphology of both Cacna1h M1560V/+ and Cacna1h -/- mice was normal. Cacna1h M1560V/+ mice had elevated aldosterone:renin ratios (a screening parameter for primary aldosteronism). Their adrenal Cyp11b2 (aldosterone synthase) expression was increased and remained elevated on a high-salt diet (relative autonomy, characteristic of primary aldosteronism), but plasma aldosterone was only elevated in male animals. The systolic blood pressure of Cacna1h M1560V/+ mice was 8 mmHg higher than in wild-type littermates and remained elevated on a high-salt diet. Cacna1h -/- mice had elevated renal Ren1 (renin-1) expression but normal adrenal Cyp11b2 levels, suggesting that in the absence of CaV3.2, stimulation of the renin-angiotensin system activates alternative calcium entry pathways to maintain normal aldosterone production. On a cellular level, Cacna1h M1560V/+ adrenal slices showed increased baseline and peak intracellular calcium concentrations in the zona glomerulosa compared to controls, but the frequency of calcium spikes did not rise. We conclude that FH-IV, on a molecular level, is caused by elevated intracellular Ca2+ concentrations as a signal for aldosterone production in adrenal glomerulosa cells. We demonstrate that a germline Cacna1h gain-of-function mutation is sufficient to cause mild primary aldosteronism, whereas loss of CaV3.2 channel function can be compensated for in a chronic setting.

Single-cell dissection of cellular components and interactions shaping the tumor immune phenotypes in ovarian cancer

Cancer cell

2021 Apr 27

Hornburg, M;Desbois, M;Lu, S;Guan, Y;Lo, AA;Kaufman, S;Elrod, A;Lotstein, A;DesRochers, TM;Munoz-Rodriguez, JL;Wang, X;Giltnane, J;Mayba, O;Turley, SJ;Bourgon, R;Daemen, A;Wang, Y;
PMID: 33961783 | DOI: 10.1016/j.ccell.2021.04.004

Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.

Host IL11 Signaling Suppresses CD4+ T cell-Mediated Antitumor Responses to Colon Cancer in Mice

Cancer immunology research

2021 Apr 27

Huynh, J;Baloyan, D;Chisanga, D;Shi, W;O'Brien, M;Afshar-Sterle, S;Alorro, M;Pang, L;Williams, DS;Parslow, AC;Thilakasiri, P;Eissmann, MF;Boon, L;Masson, F;Chand, AL;Ernst, M;
PMID: 33906864 | DOI: 10.1158/2326-6066.CIR-19-1023

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell-mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell-extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related commentary by van der Burg.

The Great Deceiver: miR-2392's Hidden Role in Driving SARS-CoV-2 Infection

bioRxiv : the preprint server for biology

2021 Apr 27

McDonald, JT;Enguita, FJ;Taylor, D;Griffin, RJ;Priebe, W;Emmett, MR;McGrath, M;Sajadi, MM;Harris, AD;Clement, J;Dybas, JM;Aykin-Burns, N;Guarnieri, JW;Singh, LN;Grabham, P;Baylin, SB;Yousey, A;Pearson, AN;Corry, PM;Saravia-Butler, A;Aunins, TR;Nagpal, P;Meydan, C;Foox, J;Mozsary, C;Cerqueira, B;Zaksas, V;Singh, U;Wurtele, ES;Costes, SV;Galeano, D;Paccanaro, A;Meinig, SL;Hagan, RS;Bowman, NM;UNC COVID-19 Pathobiology Consortium, ;Wolfgang, MC;Altinok, S;Sapoval, N;Treangen, TJ;Frieman, M;Vanderburg, C;Wallace, DC;Schisler, J;Mason, CE;Chatterjee, A;Meller, R;Beheshti, A;
PMID: 33948587 | DOI: 10.1101/2021.04.23.441024

MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation that have a major impact on many diseases and provides an exciting avenue towards antiviral therapeutics. From patient transcriptomic data, we have discovered a circulating miRNA, miR-2392, that is directly involved with SARS-CoV-2 machinery during host infection. Specifically, we found that miR-2392 was key in driving downstream suppression of mitochondrial gene expression, increasing inflammation, glycolysis, and hypoxia as well as promoting many symptoms associated with COVID-19 infection. We demonstrate miR-2392 is present in the blood and urine of COVID-19 patients tested, but not detected in COVID-19 negative patients. These findings indicate the potential for developing a novel, minimally invasive, COVID-19 detection method. Lastly, using both in vitro human and in vivo hamster models, we have developed a novel miRNA-based antiviral therapeutic targeting miR-2392 that significantly reduces SARS-CoV-2 viability and may potentially inhibit a COVID-19 disease state in the host.

Pharmacological HIF-PHD inhibition reduces renovascular resistance and increases glomerular filtration by stimulating nitric oxide generation

Acta physiologica (Oxford, England)

2021 Apr 26

Burmakin, M;Fasching, A;Kobayashi, H;Urrutia, AA;Damdimopoulos, A;Palm, F;Haase, VH;
PMID: 33900001 | DOI: 10.1111/apha.13668

Hypoxia-inducible factors (HIFs) are O2 -sensitive transcription factors that regulate multiple biological processes which are essential for cellular adaptation to hypoxia. Small molecule inhibitors of HIF-prolyl hydroxylase domain (PHD) dioxygenases (HIF-PHIs) activate HIF-dependent transcriptional programs and have broad clinical potential. HIF-PHIs are currently in global late-stage clinical development for the treatment of anaemia associated with chronic kidney disease. Although the effects of hypoxia on renal haemodynamics and function have been studied in animal models and in humans living at high altitude, the effects of pharmacological HIF activation on renal haemodynamics, O2 metabolism and metabolic efficiency are not well understood. Using a cross-sectional study design, we investigated renal haemodynamics, O2 metabolism, gene expression and NO production in healthy rats treated with different doses of HIF-PHIs roxadustat or molidustat compared to vehicle control. Systemic administration of roxadustat or molidustat resulted in a dose-dependent reduction in renovascular resistance (RVR). This was associated with increased glomerular filtration rate (GFR), urine flow and tubular sodium transport rate (TNa ). Although both total O2 delivery and TNa were increased, more O2 was extracted per transported sodium in rats treated with high-doses of HIF-PHIs, suggesting a reduction in metabolic efficiency. Changes in RVR and GFR were associated with increased nitric oxide (NO) generation and substantially suppressed by pharmacological inhibition of NO synthesis. Our data provide mechanistic insights into dose-dependent effects of short-term pharmacological HIF activation on renal haemodynamics, glomerular filtration and O2 metabolism and identify NO as a major mediator of these effects.

Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH

Analytical biochemistry

2021 Apr 26

Chen, K;Wang, M;Zhang, R;Li, J;
PMID: 33915117 | DOI: 10.1016/j.ab.2021.114211

Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBER in situ hybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediated in situ RNA imaging tool-RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.

Randomised trial of radiotherapy with weekly cisplatin or cetuximab in low risk HPV associated oropharyngeal cancer (TROG 12.01)- a Trans-Tasman Radiation Oncology Group study

International journal of radiation oncology, biology, physics

2021 Apr 24

Rischin, D;King, M;Kenny, L;Porceddu, S;Wratten, C;Macann, A;Jackson, JE;Bressel, M;Herschtal, A;Fisher, R;Fua, T;Lin, C;Liu, C;Hughes, BGM;McGrath, M;McDowell, L;Corry, J;
PMID: 34098030 | DOI: 10.1016/j.ijrobp.2021.04.015

The excellent prognosis of patients with low risk HPV associated oropharyngeal squamous cell carcinoma has led to concerns about overtreatment and excessive toxicity with radiotherapy and cisplatin, leading to interest in de-intensification trials. We investigated whether cetuximab, an EGFR targeting antibody, when combined with radiotherapy would result in a decrease in symptom burden and toxicity with similar efficacy when compared to weekly cisplatin.XXXX, a randomised, multicentre trial involving 15 sites in XXXX enrolled patients with HPV associated oropharyngeal squamous cell carcinoma, AJCC 7th edition Stage III (excluding T1-2N1) or stage IV (excluding T4 and/or N3 and/or N2b-c if smoking history >10 pack years and/or distant metastases). Patients were randomised (1:1) to receive radiotherapy (70Gy in 35 fractions) with either weekly cisplatin, 7 doses of 40mg/m2 or cetuximab, loading dose of 400mg/m2 followed by 7 weekly doses of 250 mg/m2. The primary outcome was symptom severity assessed by the MD Anderson Symptom Inventory Head and Neck Symptom Severity Scale from baseline to 13 weeks post completion of radiotherapy using the area under the curve (AUC). Trial was registered on ClinicalTrials.gov: XXXX RESULTS: Between 17th June 2013 and 7th June 2018, 189 patients were enrolled, with 92 on cisplatin arm and 90 on cetuximab included in the main analysis. There was no difference in the primary endpoint of symptom severity; difference in AUC cetuximab - cisplatin was 0.05 (95%CI: -0.19, 0.30), p= 0.66. The T-score (mean number of ≥ grade 3 acute adverse events) was 4.35 (SD 2.48) in the cisplatin arm and 3.82 (SD 1.8) in the cetuximab arm, p= 0.108. The 3 -year failure-free survival rates were 93% (95% CI: 86-97%) in the cisplatin arm and 80% (95% CI: 70-87%) in the cetuximab arm (hazard ratio = 3.0 (95% CI: 1.2-7.7); p=0.015.For patients with low risk HPV associated oropharyngeal cancer, radiotherapy and cetuximab had inferior failure-free survival without improvement in symptom burden or toxicity compared to radiotherapy and weekly cisplatin. Radiotherapy and cisplatin remains the standard of care.

Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling

Nature communications

2021 Apr 23

Yin, J;Spillman, E;Cheng, ES;Short, J;Chen, Y;Lei, J;Gibbs, M;Rosenthal, JS;Sheng, C;Chen, YX;Veerasammy, K;Choetso, T;Abzalimov, R;Wang, B;Han, C;He, Y;Yuan, Q;
PMID: 33893307 | DOI: 10.1038/s41467-021-22751-7

Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, we perform combined genetic, transcriptome, and biochemical analyses in the developing Drosophila larval brain. Here we report, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, we elucidate molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment.

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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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