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Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting

Nature neuroscience

2021 May 17

Biglari, N;Gaziano, I;Schumacher, J;Radermacher, J;Paeger, L;Klemm, P;Chen, W;Corneliussen, S;Wunderlich, CM;Sue, M;Vollmar, S;Klöckener, T;Sotelo-Hitschfeld, T;Abbasloo, A;Edenhofer, F;Reimann, F;Gribble, FM;Fenselau, H;Kloppenburg, P;Wunderlich, FT;Brüning, JC;
PMID: 34002087 | DOI: 10.1038/s41593-021-00854-0

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus represent key regulators of metabolic homeostasis. Electrophysiological and single-cell sequencing experiments have revealed a remarkable degree of heterogeneity of these neurons. However, the exact molecular basis and functional consequences of this heterogeneity have not yet been addressed. Here, we have developed new mouse models in which intersectional Cre/Dre-dependent recombination allowed for successful labeling, translational profiling and functional characterization of distinct POMC neurons expressing the leptin receptor (Lepr) and glucagon like peptide 1 receptor (Glp1r). Our experiments reveal that POMCLepr+ and POMCGlp1r+ neurons represent largely nonoverlapping subpopulations with distinct basic electrophysiological properties. They exhibit a specific anatomical distribution within the arcuate nucleus and differentially express receptors for energy-state communicating hormones and neurotransmitters. Finally, we identify a differential ability of these subpopulations to suppress feeding. Collectively, we reveal a notably distinct functional microarchitecture of critical metabolism-regulatory neurons.

Increased hippocampal excitability in miR-324-null mice

Scientific reports

2021 May 17

Hayman, DJ;Modebadze, T;Charlton, S;Cheung, K;Soul, J;Lin, H;Hao, Y;Miles, CG;Tsompani, D;Jackson, RM;Briggs, MD;Piróg, KA;Clark, IM;Barter, MJ;Clowry, GJ;LeBeau, FEN;Young, DA;
PMID: 34001919 | DOI: 10.1038/s41598-021-89874-1

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3'UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses

Cells

2021 May 17

Sheat, S;Margaria, P;Winter, S;
PMID: 34067728 | DOI: 10.3390/cells10051221

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Infection by Diverse HIV-1 Subtypes Leads to Different Elevations in HERV-K Transcriptional Levels in Human T Cell Lines

Frontiers in Microbiology

2021 May 17

Li, X;Guo, Y;Li, H;Huang, X;Pei, Z;Wang, X;Liu, Y;Jia, L;Li, T;Bao, Z;Wang, X;Han, L;Han, J;Li, J;Li, L;
| DOI: 10.3389/fmicb.2021.662573

Human endogenous retroviruses (HERVs) make up ~8% of the human genome, and for millions of years, they have been subject to strict biological regulation. Many HERVs do not participate in normal physiological activities in the body. However, in some pathological conditions, they can be abnormally activated. For example, HIV infection can cause abnormal activation of HERVs, and under different infection conditions, HERV expression may be different. We observed significant differences in HERV-K transcription levels among HIV-1 subtype-infected individuals. The transcriptional levels in the HERV-K gag region were significantly increased in HIV-1 B subtype-infected patients, while the transcriptional levels in the HERV-K pol region were significantly increased in CRF01_AE and CRF07_BC subtype-infected patients. In vitro, the transcriptional levels of HEVR-K were increased 5-fold and 15-fold in MT2 cells transfected with two different HIV-1 strains (B and CRF01_AE, respectively). However, there was no significant difference in transcriptional levels among regions of HERV-K. When MT2 cells were infected with different subtypes of HIV-1 Tat proteins (B, CRF01_AE), which is constructed by lentiviruses, and the transcription levels of HERV-K were increased 4-fold and 2-fold, respectively. Thus, different subtypes of HIV-1 have different effects on HERV-K transcription levels, which may be caused by many factors, not only Tat protein.

Charting human development using a multi-endodermal organ atlas and organoid models

Cell

2021 May 17

Yu, Q;Kilik, U;Holloway, EM;Tsai, YH;Harmel, C;Wu, A;Wu, JH;Czerwinski, M;Childs, CJ;He, Z;Capeling, MM;Huang, S;Glass, IA;Higgins, PDR;Treutlein, B;Spence, JR;Camp, JG;
PMID: 34019796 | DOI: 10.1016/j.cell.2021.04.028

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Single cell RNA-seq analysis of the flexor digitorum brevis mouse myofibers

Skeletal muscle

2021 May 17

Verma, RX;Kannan, S;Lin, BL;Fomchenko, KM;Nieuwenhuis, TO;Patil, AH;Lukban, C;Yang, X;Fox-Talbot, K;McCall, MN;Kwon, C;Kass, DA;Rosenberg, AZ;Halushka, MK;
PMID: 34001262 | DOI: 10.1186/s13395-021-00269-2

Skeletal muscle myofibers can be separated into functionally distinct cell types that differ in gene and protein expression. Current single cell expression data is generally based upon single nucleus RNA, rather than whole myofiber material. We examined if a whole-cell flow sorting approach could be applied to perform single cell RNA-seq (scRNA-seq) in a single muscle type. We performed deep, whole cell, scRNA-seq on intact and fragmented skeletal myofibers from the mouse fast-twitch flexor digitorum brevis muscle utilizing a flow-gated method of large cell isolation. We performed deep sequencing of 763 intact and fragmented myofibers. Quality control metrics across the different gates indicated only 171 of these cells were optimal, with a median read count of 239,252 and an average of 12,098 transcripts per cell. scRNA-seq identified three clusters of myofibers (a slow/fast 2A cluster and two fast 2X clusters). Comparison to a public skeletal nuclear RNA-seq dataset demonstrated a diversity in transcript abundance by method. RISH validated multiple genes across fast and slow twitch skeletal muscle types. This study introduces and validates a method to isolate intact skeletal muscle myofibers to generate deep expression patterns and expands the known repertoire of fiber-type-specific genes.

CCL21 activation of the MALAT1/SRSF1/mTOR axis underpins the development of gastric carcinoma

Journal of translational medicine

2021 May 17

Fu, Q;Tan, X;Tang, H;Liu, J;
PMID: 34001131 | DOI: 10.1186/s12967-021-02806-5

As a significant cause of malignancy mortality, gastric carcinoma (GC) has been well documented to be an often-fatal diagnosis. Despite the limitations of effective therapy, immunotherapy has emerged as a promising therapeutic approach capable of killing cancer cells via the immune system. The current study was conducted to investigate the effect of cytokine C-C motif chemokine ligand 21 (CCL21) on GC progression through the metastasis-associated lung adenocarcinoma transcript 1/serine arginine-rich splicing factor 1/mammalian target of rapamycin (MALAT1/SRSF1/mTOR) axis. Bioinformatics analysis was conducted to identify the key genes associated with GC and to subsequently predict their downstream genes. The effect of CCL21, MALAT1, and SRSF1 on the malignant phenotypes and epithelial-mesenchymal transition (EMT) of SGC-7901 and MGC-803 cells in-vitro and the tumorigenesis of SGC-7901 and MGC-803 cells in-vivo were assessed by expression determination and plasmid transfection. Additionally, RNA pull-down and RNA binding protein immunoprecipitation experiments were performed to determine the MALAT1-microRNA-202-3p (miR-203-3p) interaction and miR-202-3p-SRSF1 interaction followed by the analysis of their effect on the mTOR pathway. CCL21 was identified as a key GC immune gene. Overexpressed CCL21, MALAT1, and SRSF1 along with poorly expressed miR-202-3p were identified in the GC cells. CCL21 induced the MALAT1 expression in a time- and dose-dependent manner. Functionally, MALAT1 targeted miR-202-3p but upregulated SRSF1 and activated mTOR. Crucially, evidence was obtained indicating that CCL21 promoted both the malignant phenotypes and EMT of SGC-7901 and MGC-803 cells in-vitro and the tumorigenesis of SGC-7901 and MGC-803 cells in-vivo by increasing the MALAT1-induced upregulation of SRSF1. Taken together, the key observations of our study provide evidence that CCL21 enhances the progression of GC via the MALAT1/SRSF1/mTOR axis, providing a novel therapeutic target for the treatment of GC.

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes

PloS one

2021 May 17

Ademi, H;Shinde, DA;Gassmann, M;Gerst, D;Chaachouay, H;Vogel, J;Gorr, TA;
PMID: 33999935 | DOI: 10.1371/journal.pone.0251765

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin , AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.

Hox11 expression characterizes developing zeugopod synovial joints and is coupled to postnatal articular cartilage morphogenesis into functional zones in mice

Developmental biology

2021 May 16

Rux, D;Helbig, K;Koyama, E;Pacifici, M;
PMID: 34010606 | DOI: 10.1016/j.ydbio.2021.05.007

Previous studies on mouse embryo limbs have established that interzone mesenchymal progenitor cells emerging at each prescribed joint site give rise to joint tissues over fetal time. These incipient tissues undergo structural maturation and morphogenesis postnatally, but underlying mechanisms of regulation remain unknown. Hox11 genes dictate overall zeugopod musculoskeletal patterning and skeletal element identities during development. Here we asked where these master regulators are expressed in developing limb joints and whether they are maintained during postnatal zeugopod joint morphogenesis. We found that Hoxa11 was predominantly expressed and restricted to incipient wrist and ankle joints in E13.5 mouse embryos, and became apparent in medial and central regions of knees by E14.5, though remaining continuously dormant in elbow joints. Closer examination revealed that Hoxa11 initially characterized interzone and neighboring cells and was then restricted to nascent articular cartilage, intra joint ligaments and structures such as meniscal horns over prenatal time. Postnatally, articular cartilage progresses from a nondescript cell-rich, matrix-poor tissue to a highly structured, thick, zonal and mechanically competent tissue with chondrocyte columns over time, most evident at sites such as the tibial plateau. Indeed, Hox11 expression (primarily Hoxa11) was intimately coupled to such morphogenetic processes and, in particular, to the topographical rearrangement of chondrocytes into columns within the intermediate and deep zones of tibial plateau that normally endures maximal mechanical loads. Revealingly, these expression patterns were maintained even at 6 months of age. In sum, our data indicate that Hox11 genes remain engaged well beyond embryonic synovial joint patterning and are specifically tied to postnatal articular cartilage morphogenesis into a zonal and resilient tissue. The data demonstrate that Hox11 genes characterize adult, terminally differentiated, articular chondrocytes and maintain region-specificity established in the embryo.

Calcifying nested stromal-epithelial tumor: a clinicopathologic and molecular genetic study of eight cases highlighting metastatic potential and recurrent CTNNB1 and TERT promoter alterations

Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

2021 May 16

Papke, DJ;Dong, F;Zhang, X;Kozielski, R;Basturk, O;Fletcher, CDM;Zhao, L;
PMID: 33994539 | DOI: 10.1038/s41379-021-00822-w

Calcifying nested stromal-epithelial tumor (CNSET) is a rare hepatic tumor that occurs in children and young adults. With <40 cases in the literature, the mechanism for tumorigenesis and the biological behavior of CNSET remain uncertain. Here, we studied the clinicopathologic and molecular genetic features of eight CNSETs. Six patients (75%) were female, and the median age at presentation was 22.5 years (range 14-34 years). The median tumor size was 14 cm (range 2.7-18 cm). All tumors had fibrous stroma that contained organoid nests of epithelioid to spindled tumor cells with moderate amounts of palely eosinophilic cytoplasm and ovoid, vesicular nuclei. Five tumors showed calcifications, and one showed lymphovascular invasion. Necrosis was absent in all. Immunohistochemistry demonstrated nuclear β-catenin expression in five of five tested tumors and focal to diffuse nuclear WT-1 positivity in five of seven. Hepatocellular markers (HepPar-1, arginase-1, and albumin in situ hybridization) and neuroendocrine markers (synaptophysin, chromogranin, and INSM1) were uniformly negative. Next-generation sequencing demonstrated CTNNB1 alterations in all seven sequenced tumors. Sanger sequencing demonstrated TERT promoter mutations in all six sequenced tumors. Clinical follow-up was available for seven patients (median duration 4.4 years; range 1.2-6.2 years): four (57%) developed metastatic disease; all four developed lung metastases; and two also had abdominal metastases. All four patients with metastatic disease also had persistent or recurrent liver tumors. Three patients with metastases were alive with disease at the most recent follow-up and one died of disease. The other three patients with available follow-up did not develop metastasis or recurrence. One tumor treated with neoadjuvant chemotherapy showed no response, and another showed 90% tumor fibrosis; the latter patient remained disease-free at 6.2 years of follow-up. Our series demonstrates the presence of TERT promoter mutations and CTNNB1 alterations in all sequenced tumors and suggests that CNSET might perhaps be more aggressive than previously reported.

Is thyroid gland a target of SARS-CoV-2 infection? Results of the analysis of necropsy thyroid specimens from COVID-19 patients

Endocrine Abstracts

2021 May 15

Macedo, S;Pestana, A;Liliana, R;Neves, C;Susana, G;Guimarães, A;Dolhnikoff, M;Saldiva, P;Carneiro, F;Sobrinho-Simões, M;Soares, P;
| DOI: 10.1530/endoabs.73.oc14.3

In the 2002 outbreak of severe acute respiratory syndrome (SARS) a number of patients presented abnormalities in the thyroid functioning, neuroendocrine and calcium homeostasis. It was detected in autopsies from SARS Coronavirus (SARS-CoV) patients that the thyroid gland was significantly affected by the disease, with extensive injury and death of follicular and parafollicular cells. In the present SARS-CoV-2 pandemic some studies start to report acute thyroiditis and alterations in the levels of thyroid hormones [(triiodothyronine (T3), thyroxine (T4), thyroid stimulating hormone (TSH)]. Thyroid cells present high levels of mRNA expression of angiotensin-converting enzyme 2 (ACE2), the host receptor for SARS-CoV-2. It remains poorly studied the thyroid expression of proteins that predispose to SARS-CoV-2 infection and if thyroid cells can be a direct or indirect target of SARS-CoV-2 infection.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus

Development (Cambridge, England)

2021 May 15

Kaiser, K;Jang, A;Kompanikova, P;Lun, MP;Prochazka, J;Machon, O;Dani, N;Prochazkova, M;Laurent, B;Gyllborg, D;van Amerongen, R;Fame, RM;Gupta, S;Wu, F;Barker, RA;Bukova, I;Sedlacek, R;Kozmik, Z;Arenas, E;Lehtinen, MK;Bryja, V;
PMID: 34032267 | DOI: 10.1242/dev.192054

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

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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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