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Photothermal mediated rolling circle amplification toward specific and direct in situ mRNA detection

Biosensors & bioelectronics

2021 Jul 21

Liu, D;Li, W;Yang, M;Qiu, L;Pian, H;Huang, Y;Chen, M;Zheng, Z;
PMID: 34330037 | DOI: 10.1016/j.bios.2021.113507

Rolling circle amplification (RCA) had the prospect of assisting clinic diagnosis with advantage in in situ mRNA detection at single cell level. However, for direct mRNA detection, RCA had relatively low detection specificity and efficiency. Here, we introduced 4-(10, 15, 20-Triphenylporphyrin-5-yl)phenylamine (TPP) modified Au nanoparticle (Au-TPP) to improve the specificity of in-situ RCA. Through photothermal effect, Au-TPP acted as the specific heat source upon irradiation of 635 nm laser. The photothermal mediated RCA would be initiated only when the Au-TPP as well as the padlock anchored adjacently on the same target mRNA. Furthermore, we introduced 'C' form target-specific oligonucleotide linker probes to make generic padlock and Au-TPP for different mRNA targets, so that for a new mRNA target one does not have to redesign the padlock and the Au-TPP probe. By these strategies, we successfully developed a specific and photothermal mediated hyperbranched rolling circle amplification for direct in situ mRNA detection, suitable for both formalin-fixed paraffin-embedded (FFPE) tissue section and frozen tissue section.

Compartmentalization of interleukin 36 subfamily according to inducible and constitutive expression in the kidneys of a murine autoimmune nephritis model

Cell and tissue research

2021 Jul 21

Namba, T;Ichii, O;Nakamura, T;Masum, MA;Otani, Y;Hosotani, M;Elewa, YHA;Kon, Y;
PMID: 34287716 | DOI: 10.1007/s00441-021-03495-8

The interleukin (IL) 36 subfamily belongs to the IL-1 family and is comprised of agonists (IL-36α, IL-36β, IL-36γ) and antagonists (IL-36Ra, IL-38). We previously reported IL-36α overexpression in renal tubules of chronic nephritis mice. To understand the localization status and biological relationships among each member of the IL-36 subfamily in the kidneys, MRL/MpJ-Faslpr/lpr mice were investigated as autoimmune nephritis models using pathology-based techniques. MRL/MpJ-Faslpr/lpr mice exhibited disease onset from 3 months and severe nephritis at 6-7 months (early and late stages, respectively). Briefly, IL-36γ and IL-36Ra were constitutively expressed in murine kidneys, while the expression of IL-36α, IL-36β, IL-36Ra, and IL-38 was induced in MRL/MpJ-Faslpr/lpr mice. IL-36α expression was significantly increased and localized to injured tubular epithelial cells (TECs). CD44+-activated parietal epithelial cells (PECs) also exhibited higher IL-36α-positive rates, particularly in males. IL-36β and IL-38 are expressed in interstitial plasma cells. Quantitative indices for IL-36α and IL-38 positively correlated with nephritis severity. Similar to IL-36α, IL-36Ra localized to TECs and PECs at the late stage; however, MRL/MpJ-Faslpr/lpr and healthy MRL/MpJ mice possessed IL-36Ra+ smooth muscle cells in kidney arterial tunica media at both stages. IL-36γ was constitutively expressed in renal sympathetic axons regardless of strain and stage. IL-36 receptor gene was ubiquitously expressed in the kidneys and was induced proportional to disease severity. MRL/MpJ-Faslpr/lpr mice kidneys possessed significantly upregulated IL-36 downstream candidates, including NF-κB- or MAPK-pathway organizing molecules. Thus, the IL-36 subfamily contributes to homeostasis and inflammation in the kidneys, and especially, an IL-36α-dominant imbalance could strongly impact nephritis deterioration.

Increased gene expression variability in BRCA1-associated and basal-like breast tumours

Breast cancer research and treatment

2021 Jul 21

Wiggins, GAR;Black, MA;Dunbier, A;Morley-Bunker, AE;kConFab Investigators, ;Pearson, JF;Walker, LC;
PMID: 34287743 | DOI: 10.1007/s10549-021-06328-y

Inherited variants in the cancer susceptibility genes, BRCA1 and BRCA2 account for up to 5% of breast cancers. Multiple gene expression studies have analysed gene expression patterns that maybe associated with BRCA12 pathogenic variant status; however, results from these studies lack consensus. These studies have focused on the differences in population means to identified genes associated with BRCA1/2-carriers with little consideration for gene expression variability, which is also under genetic control and is a feature of cellular function.We measured differential gene expression variability in three of the largest familial breast cancer datasets and a 2116 breast cancer meta-cohort. Additionally, we used RNA in situ hybridisation to confirm expression variability of EN1 in an independent cohort of more than 500 breast tumours.BRCA1-associated breast tumours exhibited a 22.8% (95% CI 22.3-23.2) increase in transcriptome-wide gene expression variability compared to BRCAx tumours. Additionally, 40 genes were associated with BRCA1-related breast cancers that had ChIP-seq data suggestive of enriched EZH2 binding. Of these, two genes (EN1 and IGF2BP3) were significantly variable in both BRCA1-associated and basal-like breast tumours. RNA in situ analysis of EN1 supported a significant (p = 6.3 × 10-04) increase in expression variability in BRCA1-associated breast tumours.Our novel results describe a state of increased gene expression variability in BRCA1-related and basal-like breast tumours. Furthermore, genes with increased variability may be driven by changes in DNA occupancy of epigenetic effectors. The variation in gene expression is replicable and led to the identification of novel associations between genes and disease phenotypes.

miR-29a-3p inhibits endometrial cancer cell proliferation, migration and invasion by targeting VEGFA/CD C42/PAK1

BMC cancer

2021 Jul 21

Geng, A;Luo, L;Ren, F;Zhang, L;Zhou, H;Gao, X;
PMID: 34289832 | DOI: 10.1186/s12885-021-08506-z

This study aimed to investigate the mechanism of miR-29a-3p in regulating endometrial cancer (EC) progression.A total of 72 EC patients were enrolled. EC cells were transfected. Cells proliferation, cloning ability, migration and invasion were researched by MTT assay, colony formation experiment, cell scratch test and Transwell experiment respectively. Dual-luciferase reporter assay was performed. Xenograft experiment was conducted using nude mice. miR-29a-3p, VEGFA, CDC42, PAK1 and p-PAK1 expression in cells/tissues was investigated by qRT-PCR and Western blot.miR-29a-3p expression was aberrantly reduced in EC patients, which was associated with poor outcome. miR-29a-3p inhibited EC cells proliferation, cloning formation, migration and invasion (P < 0.05 or P < 0.01 or P < 0.001). miR-29a-3p inhibited CDC42/PAK1 signaling pathway activity in EC cells (P < 0.01). VEGFA expression was directly inhibited by miR-29a-3p. miR-29a-3p suppressed EC cells malignant phenotype in vitro and growth in vivo by targeting VEGFA/CDC42/PAK1 signaling pathway (P < 0.05 or P < 0.01).miR-29a-3p inhibits EC cells proliferation, migration and invasion by targeting VEGFA/CDC42/PAK1 signaling pathway.

Gα15 in early onset of pancreatic ductal adenocarcinoma

Scientific reports

2021 Jul 21

Innamorati, G;Wilkie, TM;Malpeli, G;Paiella, S;Grasso, S;Rusev, B;Leone, BE;Valenti, MT;Carbonare, LD;Cheri, S;Giacomazzi, A;Zanotto, M;Guardini, V;Deiana, M;Zipeto, D;Serena, M;Parenti, M;Guzzi, F;Lawlor, RT;Malerba, G;Mori, A;Malleo, G;Giacomello, L;Salvia, R;Bassi, C;
PMID: 34290274 | DOI: 10.1038/s41598-021-94150-3

The GNA15 gene is ectopically expressed in human pancreatic ductal adenocarcinoma cancer cells. The encoded Gα15 protein can promiscuously redirect GPCR signaling toward pathways with oncogenic potential. We sought to describe the distribution of GNA15 in adenocarcinoma from human pancreatic specimens and to analyze the mechanism driving abnormal expression and the consequences on signaling and clinical follow-up. We detected GNA15 expression in pre-neoplastic pancreatic lesions and throughout progression. The analysis of biological data sets, primary and xenografted human tumor samples, and clinical follow-up shows that elevated expression is associated with poor prognosis for GNA15, but not any other GNA gene. Demethylation of the 5' GNA15 promoter region was associated with ectopic expression of Gα15 in pancreatic neoplastic cells, but not in adjacent dysplastic or non-transformed tissue. Down-modulation of Gα15 by shRNA or CRISPR/Cas9 affected oncogenic signaling, and reduced adenocarcimoma cell motility and invasiveness. We conclude that de novo expression of wild-type GNA15 characterizes transformed pancreatic cells. The methylation pattern of GNA15 changes in preneoplastic lesions coincident with the release a transcriptional blockade that allows ectopic expression to persist throughout PDAC progression. Elevated GNA15 mRNA correlates with poor prognosis. In addition, ectopic Gα15 signaling provides an unprecedented mechanism in the early steps of pancreas carcinogenesis distinct from classical G protein oncogenic mutations described previously in GNAS and GNAQ/GNA11.

Myomixer is expressed during embryonic and post-larval hyperplasia, muscle regeneration and differentiation of myoblats in rainbow trout (Oncorhynchus mykiss)

Gene

2021 Jul 20

Perello-Amoros, M;Rallière, C;Gutiérrez, J;Gabillard, JC;
PMID: 33961974 | DOI: 10.1016/j.gene.2021.145688

In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.

Npas4 regulates IQSEC3 expression in hippocampal somatostatin interneurons to mediate anxiety-like behavior

Cell reports

2021 Jul 20

Kim, S;Park, D;Kim, J;Kim, D;Kim, H;Mori, T;Jung, H;Lee, D;Hong, S;Jeon, J;Tabuchi, K;Cheong, E;Kim, J;Um, JW;Ko, J;
PMID: 34289353 | DOI: 10.1016/j.celrep.2021.109417

Activity-dependent GABAergic synapse plasticity is important for normal brain functions, but the underlying molecular mechanisms remain incompletely understood. Here, we show that Npas4 (neuronal PAS-domain protein 4) transcriptionally regulates the expression of IQSEC3, a GABAergic synapse-specific guanine nucleotide-exchange factor for ADP-ribosylation factor (ARF-GEF) that directly interacts with gephyrin. Neuronal activation by an enriched environment induces Npas4-mediated upregulation of IQSEC3 protein specifically in CA1 stratum oriens layer somatostatin (SST)-expressing GABAergic interneurons. SST+ interneuron-specific knockout (KO) of Npas4 compromises synaptic transmission in these GABAergic interneurons, increases neuronal activity in CA1 pyramidal neurons, and reduces anxiety behavior, all of which are normalized by the expression of wild-type IQSEC3, but not a dominant-negative ARF-GEF-inactive mutant, in SST+ interneurons of Npas4-KO mice. Our results suggest that IQSEC3 is a key GABAergic synapse component that is directed by Npas4 and ARF activity, specifically in SST+ interneurons, to orchestrate excitation-to-inhibition balance and control anxiety-like behavior.

TRESK channel contributes to depolarization-induced shunting inhibition and modulates epileptic seizures

Cell reports

2021 Jul 20

Huang, W;Ke, Y;Zhu, J;Liu, S;Cong, J;Ye, H;Guo, Y;Wang, K;Zhang, Z;Meng, W;Gao, TM;Luhmann, HJ;Kilb, W;Chen, R;
PMID: 34289346 | DOI: 10.1016/j.celrep.2021.109404

Glutamatergic and GABAergic synaptic transmission controls excitation and inhibition of postsynaptic neurons, whereas activity of ion channels modulates neuronal intrinsic excitability. However, it is unclear how excessive neuronal excitation affects intrinsic inhibition to regain homeostatic stability under physiological or pathophysiological conditions. Here, we report that a seizure-like sustained depolarization can induce short-term inhibition of hippocampal CA3 neurons via a mechanism of membrane shunting. This depolarization-induced shunting inhibition (DShI) mediates a non-synaptic, but neuronal intrinsic, short-term plasticity that is able to suppress action potential generation and postsynaptic responses by activated ionotropic receptors. We demonstrate that the TRESK channel significantly contributes to DShI. Disruption of DShI by genetic knockout of TRESK exacerbates the sensitivity and severity of epileptic seizures of mice, whereas overexpression of TRESK attenuates seizures. In summary, these results uncover a type of homeostatic intrinsic plasticity and its underlying mechanism. TRESK might represent a therapeutic target for antiepileptic drugs.

A Novel Model for Papillomavirus-Mediated Anal Disease and Cancer Using the Mouse Papillomavirus

mBio

2021 Jul 20

Blaine-Sauer, S;Shin, MK;Matkowskyj, KA;Ward-Shaw, E;Lambert, PF;
PMID: 34281391 | DOI: 10.1128/mBio.01611-21

Up to 95% of all anal cancers are associated with infection by human papillomavirus (HPV); however, no established preclinical model exists for high-grade anal disease and cancer mediated by a natural papillomavirus infection. To establish an infection-mediated model, we infected both immunocompromised NSG and immunocompetent FVB/NJ mice with the recently discovered murine papillomavirus MmuPV1, with and without the additional cofactors of UV B radiation (UVB) and/or the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Infections were tracked via lavages and swabs for MmuPV1 DNA, and pathology was assessed at the endpoint. Tissues were analyzed for biomarkers of viral infection and papillomavirus-mediated disease, and the localization of viral infection was investigated using biomarkers to characterize the anal microanatomical zones. IMPORTANCE We show, for the first time, that MmuPV1 infection is sufficient to efficiently mediate high-grade squamous intraepithelial lesions in the anal tract of mice using the NSG immunocompromised strain and that MmuPV1, in combination with the chemical carcinogen DMBA, has carcinogenic potential. We further show that MmuPV1 is able to persist for up to 6 months in the anal tract of FVB/NJ mice irradiated with UVB and contributes to high-grade disease and cancer in an immunocompetent strain. We demonstrate that MmuPV1 preferentially localizes to the anal transition zone and that this localization is not an artifact of infection methodology. This study presents a valuable new preclinical model for studying papillomavirus-mediated anal disease driven by a natural infection.

Adventitial Microcirculation Is a Major Target of SARS-CoV-2-Mediated Vascular Inflammation

Biomolecules

2021 Jul 20

Vasuri, F;Ciavarella, C;Collura, S;Mascoli, C;Valente, S;Degiovanni, A;Gargiulo, M;Capri, M;Pasquinelli, G;
| DOI: 10.3390/biom11071063

We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

Inhibin-positive hepatic carcinoma: proposal for a solid-tubulocystic variant of intrahepatic cholangiocarcinoma

Human pathology

2021 Jul 20

Wen, KW;Joseph, NM;Srivastava, A;Saunders, TA;Jain, D;Rank, J;Feely, M;Zarrinpar, A;Al Diffalha, S;Shyn, PB;Graham, RP;Drage, MG;Kakar, S;
PMID: 34298064 | DOI: 10.1016/j.humpath.2021.07.004

Inhibin-positive hepatic carcinoma is a rare primary liver neoplasm that resembles sex cord stromal tumor and thyroid follicular tumors. The term "cholangioblastic variant of intrahepatic cholangiocarcinoma" has been proposed. This study describes the clinicopathologic, immunophenotypic, and molecular features of a small series (n=6) of this rare tumor. Albumin in situ hybridization (ISH) and capture-based next-generation sequencing (NGS) were also performed. All tumors occurred in young women (mean age 32.5 years, range 19-44 years) as a solitary large mass (mean 15.8 cm, range 6.9-23.5 cm). All tumors showed a highly distinctive morphology with sheets and large nests of tumor cells alternating with tubular and cystic areas imparting a sex cord-like or thyroid follicle-like morphology. Cytologic atypia was mild and mitotic activity was low. All cases were positive for inhibin, as well as pancytokeratin, CK7, CK19 and albumin in situ hybridization. Synaptophysin and chromogranin showed focal or patchy staining, while INSM1 was negative. Markers for hepatocellular differentiation, thyroid origin and sex cord stromal tumor were negative. There were no recurrent genomic changes based on capture-based next-generation sequencing (NGS) of ∼ 500 cancer genes. Recurrence and/or metastasis was seen in 3 (50%) cases (follow-up time range for all cases: 5 months-2 years). In conclusion, this series describes the distinctive morphology, immunophenotypic features and diffuse albumin staining in 6 cases of a rare inhibin-positive primary liver carcinoma that runs an aggressive course similar to intrahepatic cholangiocarcinoma. Genomic changes typical of cholangiocarcinoma or hepatocellular carcinoma were not identified, and there were no recurrent genetic abnormalities. We propose the term "solid-tubulocystic variant of intrahepatic cholangiocarcinoma" to reflect the spectrum of morphologic patterns observed in this tumor.

SARS-CoV2 infects pancreatic beta cells in vivo and induces cellular and subcellular disruptions that reflect beta cell dysfunction

Research square

2021 Jul 20

Millette, K;Cuala, J;Wang, P;Marks, C;Woo, V;Hayun, M;Kang, H;Martin, M;Dhawan, S;Chao, L;Fraser, S;Junge, J;Lewis, M;Georgia, S;
PMID: 34312617 | DOI: 10.21203/rs.3.rs-592374/v1

Increasing evidence of new-onset diabetes during the COVID19 pandemic indicates that the SARS-CoV2 virus may drive beta-cell dysfunction leading to diabetes, but it is unclear if it is a primary or secondary effect. Here, we present evidence of SARS-CoV-2 infection of pancreatic beta cells in vivo using a robust and reproducible non-human primates model of mild to moderate COVID19 pathogenesis. Pancreas from SARS-CoV-2 infected subjects were positive for the SARS-CoV2 spike protein by immunohistochemistry and structures indicative of viral replication were evident by electron microscopy. Total beta cell area was decreased in SARS-CoV-2-infected pancreas, attributable to beta cell atrophy. Beta cell granularity was decreased. These histologic phenotypes persisted beyond the duration of the clinical disease course. Detailed electron microscopy of SARS-CoV-2 infected beta-cells revealed ultrastructural hallmarks of beta cell stress that are seen in islets of patients with Type 2 diabetes, including disrupted mitochondria and dilated endoplasmic reticulum. To assess the metabolic status of beta cells from SARS-CoV-2-infected subjects, we used fluorescence life-time imaging to measure the ratio of free and bound NADH as a surrogate of glycolytic and oxidative metabolism. We report an increase in free NADH levels, suggesting that beta cells from SARS-CoV-2-infected subjects adopt a more glycolytic metabolic profile. Taken together, we conclude that SARS-CoV-2 infection induces beta cell stress that may compromise beta-cell function beyond the duration of the disease course. This raises the possibility that the beta cell stress and injury may have clinical implications of the long-term future health of patients that have recovered from COVID19.

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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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Advanced Cell Diagnostics

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