Identifying Novel Genes and Variants in Immune and Coagulation Pathways Associated with Macular Degeneration

Ophthalmology Science

2022 Aug 01

Huan, T;Cheng, S;Tian, B;Punzo, C;Lin, H;Daly, M;Seddon, J;
| DOI: 10.1016/j.xops.2022.100206

Purpose To select individuals and families with low genetic burden for age-related macular degeneration (AMD), to inform the clinical diagnosis of macular disorders, and to find novel genetic variants associated with macular disease in affected families. Design Genetic association study based on targeted and whole exome sequencing. Participants 758 subjects (481 individuals with maculopathy and 277 controls), including 316 individuals in 72 families. Methods We focused on 150 genes involved in the complement, coagulation, and inflammatory pathways. Single-variant tests were performed on 3062 variants shared among 5 or more subjects using logistic regression. Gene-based tests were used to evaluate aggregate effects from rare and low frequency variants (at minor allele frequency [MAF]

An mPOA-ARCAgRP pathway modulates cold-evoked eating behavior

Cell reports

2021 Aug 10

Yang, S;Tan, YL;Wu, X;Wang, J;Sun, J;Liu, A;Gan, L;Shen, B;Zhang, X;Fu, Y;Huang, J;
PMID: 34380037 | DOI: 10.1016/j.celrep.2021.109502

Enhanced appetite occurs as a means of behavioral thermoregulation at low temperature. Neural circuitry mediating this crosstalk between behavioral thermoregulation and energy homeostasis remains to be elucidated. We find that the hypothalamic orexigenic agouti-related neuropeptide (AgRP) neurons in the arcuate nucleus (ARC) are profoundly activated by cold exposure. The calcium signals in ARCAgRP neurons display an immediate-response pattern in response to cold stimulation. Cold-responsive neurons in the medial preoptic area (mPOA) make excitatory synapses onto ARCAgRP neurons. Inhibition of either ARCAgRP neurons or ARC-projecting mPOA neurons attenuates cold-evoked feeding, while activation of the mPOA-to-ARC projection increases food intake. These findings reveal an mPOA-ARCAgRP neural pathway that modulates cold-evoked feeding behavior.

ABCA1 activity in the RPE is unnecessary for RPE reverse cholesterol transport (RCT) and AMD pathophysiology

Investigative Ophthalmology & Visual Science

2023 Jan 01

Coble, M;Aranda, J;Demirs, JT;Esterberg, R;Hanks, S;Jose, S;Leehy, B;Liao, S;Niu, YZ;Qiu, Y;Yang, J;

METHODS : Gene expression of ABCA1 and ApoA1 on human donor tissue and iPSC-RPE were examined by qPCR (n=3). Bulk RNAseq examined transcript changes in key RCT genes on donor retinas across different stages of disease progression. RNAscope probes (ACDBio) were designed against abca1 transcripts with appropriate mismatch controls. Neutral lipid stain with oil-red O on 10um cryo-sections of abca1 KO and wild type (WT) eyes (N= 5). Two siRNAs knocked down abca1 in iPSC-RPE cells to assess abca1 contribution to cholesterol efflux (n=3). Samples were analyzed with the cholesterol efflux kit (ab196985) and compared to non-targeting control siRNAs. Histological analysis of ABCA1 protein using anti-ABCA1 (Invitrogen-MA516026) on human donor retinas (AMD1 vs AMD3).

An Atlas of Vagal Sensory Neurons and Their Molecular Specialization.

Cell Rep

2019 May 21

Kupari J, Häring M, Agirre E, Castelo-Branco G, Ernfors P.
PMID: 31116992 | DOI: 10.1016/j.celrep.2019.04.096

Sensory functions of the vagus nerve are critical for conscious perceptions and for monitoring visceral functions in the cardio-pulmonary and gastrointestinal systems. Here, we present a comprehensive identification, classification, and validation of the neuron types in the neural crest (jugular) and placode (nodose) derived vagal ganglia by single-cell RNA sequencing (scRNA-seq) transcriptomic analysis. Our results reveal major differences between neurons derived from different embryonic origins. Jugular neurons exhibit fundamental similarities to the somatosensory spinal neurons, including major types, such as C-low threshold mechanoreceptors (C-LTMRs), A-LTMRs, Aδ-nociceptors, and cold-, and mechano-heat C-nociceptors. In contrast, the nodose ganglion contains 18 distinct types dedicated to surveying the physiological state of the internal body. Our results reveal a vast diversity of vagal neuron types, including many previously unanticipated types, as well as proposed types that are consistent with chemoreceptors, nutrient detectors, baroreceptors, and stretch and volume mechanoreceptors of the respiratory, gastrointestinal, and cardiovascular systems.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques

Immunity

2023 May 13

Wu, HL;Busman-Sahay, K;Weber, WC;Waytashek, CM;Boyle, CD;Bateman, KB;Reed, JS;Hwang, JM;Shriver-Munsch, C;Swanson, T;Northrup, M;Armantrout, K;Price, H;Robertson-LeVay, M;Uttke, S;Kumar, MR;Fray, EJ;Taylor-Brill, S;Bondoc, S;Agnor, R;Junell, SL;Legasse, AW;Moats, C;Bochart, RM;Sciurba, J;Bimber, BN;Sullivan, MN;Dozier, B;MacAllister, RP;Hobbs, TR;Martin, LD;Panoskaltsis-Mortari, A;Colgin, LMA;Siliciano, RF;Siliciano, JD;Estes, JD;Smedley, JV;Axthelm, MK;Meyers, G;Maziarz, RT;Burwitz, BJ;Stanton, JJ;Sacha, JB;
PMID: 37236188 | DOI: 10.1016/j.immuni.2023.04.019

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

PD-L1 immunohistochemistry assay optimization to provide more comprehensive pathological information in classic Hodgkin lymphoma

Journal of Hematopathology

2023 Feb 01

Shi, Y;Mi, L;Lai, Y;Zhao, M;Jia, L;Du, T;Song, Y;Li, X;
| DOI: 10.1007/s12308-023-00530-1

Overexpression of PD-L1 can be a predictive marker for anti-PD-1 therapeutic efficacy in classic Hodgkin lymphoma (CHL); however, harmonization of different IHC assays remains to be accomplished, and interpretations of PD-L1 immunostaining results remain controversial in CHL. In this study, we sought to optimize the PD-L1 immunohistochemistry (IHC) assay in CHL. All tests were performed on a tumour tissue microarray established from 54 CHL cases. Three IHC antibodies (405.9A11, SP142, 22C3) for detecting PD-L1 expression were compared semi quantitatively with the RNAscope assay (No. 310035, ACD), and the difference in the expression in background immune cells (ICs) between assays and the associations of expression levels with densities of TILs/TAMs were also analysed. 405.9A11 demonstrated best specificity in HRS cells and best sensitivity in ICs. Positive expression of PD-L1 was more frequent in ICs (85.2%) than in HRS cells (48.1%). Different subgroups of background ICs, including tumour-associated macrophages (TAMs), were assessed and scored for CD4, CD8, FOXP3, and CD163 expression. PD-L1 expression on ICs was the factor most associated with the density of TAMs. 405.9A11 provided the most convincing PD-L1 expression results. Pathologists should report PD-L1 expression in a combined manner, including both the status of HRS cells and the percentage of PD-L1-positive ICs.

MYC-driven increases in mitochondrial DNA copy number occur early and persist throughout prostatic cancer progression

bioRxiv : the preprint server for biology

2023 Feb 21

Chen, J;Zheng, Q;Hicks, JL;Trabzonlu, L;Ozbek, B;Jones, T;Vaghasia, A;Larman, TC;Wang, R;Markowski, MC;Denmeade, SR;Pienta, KJ;Hruban, RH;Antonaraskis, ES;Gupta, A;Dang, CV;Yegnasubramanian, S;De Marzo, AM;
PMID: 36865273 | DOI: 10.1101/2023.02.20.529259

Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type-specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by two orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.

Histopathologic and proteogenomic heterogeneity reveals features of clear cell renal cell carcinoma aggressiveness

Cancer cell

2022 Dec 20

Li, Y;Lih, TM;Dhanasekaran, SM;Mannan, R;Chen, L;Cieslik, M;Wu, Y;Lu, RJ;Clark, DJ;Kołodziejczak, I;Hong, R;Chen, S;Zhao, Y;Chugh, S;Caravan, W;Naser Al Deen, N;Hosseini, N;Newton, CJ;Krug, K;Xu, Y;Cho, KC;Hu, Y;Zhang, Y;Kumar-Sinha, C;Ma, W;Calinawan, A;Wyczalkowski, MA;Wendl, MC;Wang, Y;Guo, S;Zhang, C;Le, A;Dagar, A;Hopkins, A;Cho, H;Leprevost, FDV;Jing, X;Teo, GC;Liu, W;Reimers, MA;Pachynski, R;Lazar, AJ;Chinnaiyan, AM;Van Tine, BA;Zhang, B;Rodland, KD;Getz, G;Mani, DR;Wang, P;Chen, F;Hostetter, G;Thiagarajan, M;Linehan, WM;Fenyö, D;Jewell, SD;Omenn, GS;Mehra, R;Wiznerowicz, M;Robles, AI;Mesri, M;Hiltke, T;An, E;Rodriguez, H;Chan, DW;Ricketts, CJ;Nesvizhskii, AI;Zhang, H;Ding, L;Clinical Proteomic Tumor Analysis Consortium, ;
PMID: 36563681 | DOI: 10.1016/j.ccell.2022.12.001

Clear cell renal cell carcinomas (ccRCCs) represent ∼75% of RCC cases and account for most RCC-associated deaths. Inter- and intratumoral heterogeneity (ITH) results in varying prognosis and treatment outcomes. To obtain the most comprehensive profile of ccRCC, we perform integrative histopathologic, proteogenomic, and metabolomic analyses on 305 ccRCC tumor segments and 166 paired adjacent normal tissues from 213 cases. Combining histologic and molecular profiles reveals ITH in 90% of ccRCCs, with 50% demonstrating immune signature heterogeneity. High tumor grade, along with BAP1 mutation, genome instability, increased hypermethylation, and a specific protein glycosylation signature define a high-risk disease subset, where UCHL1 expression displays prognostic value. Single-nuclei RNA sequencing of the adverse sarcomatoid and rhabdoid phenotypes uncover gene signatures and potential insights into tumor evolution. In vitro cell line studies confirm the potential of inhibiting identified phosphoproteome targets. This study molecularly stratifies aggressive histopathologic subtypes that may inform more effective treatment strategies.

Hedgehog-interacting protein acts in the habenula to regulate nicotine intake

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

2022 Nov 15

Caligiuri, SPB;Howe, WM;Wills, L;Smith, ACW;Lei, Y;Bali, P;Heyer, MP;Moen, JK;Ables, JL;Elayouby, KS;Williams, M;Fillinger, C;Oketokoun, Z;Lehmann, VE;DiFeliceantonio, AG;Johnson, PM;Beaumont, K;Sebra, RP;Ibanez-Tallon, I;Kenny, PJ;
PMID: 36346845 | DOI: 10.1073/pnas.2209870119

Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the <i>Hhip</i> gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.

Mitochondrial glutamine transporter SLC1A5_var, a potential target to suppress astrocyte reactivity in Parkinson's Disease

Cell death & disease

2022 Nov 09

Liu, Y;Cao, L;Song, Y;Kang, Z;Liu, T;Ding, J;Hu, G;Lu, M;
PMID: 36351889 | DOI: 10.1038/s41419-022-05399-z

SLC1A5 variant (SLC1A5_var) is identified as a mitochondrial glutamine transporter in cancer cells recently. However, the role of SLC1A5_var in Parkinson's disease (PD) is completely unknown. Here, we found the significant downregulation of SLC1A5_var in astrocytes and midbrain of mice treated with MPTP/MPP+ and LPS. Importantly, overexpression of SLC1A5_var ameliorated but knockdown of SLC1A5_var exacerbated MPTP/MPP+- and LPS-induced mitochondrial dysfunction. Consequently, SLC1A5_var provided beneficial effects on PD pathology including improvement of PD-like motor symptoms and rescue of dopaminergic (DA) neuron degeneration through maintaining mitochondrial energy metabolism. Moreover, SLC1A5_var reduced astrocyte reactivity via inhibition of A1 astrocyte conversion. Further investigation demonstrated that SLC1A5_var restrained the secretion of astrocytic pro-inflammatory cytokines by blunting TLR4-mediated downstream pathways. This is the first study to prove that astrocytic SLC1A5_var inhibits neuroinflammation, and rescues the loss of DA neurons and motor symptoms involved in PD progression, which provides a novel target for PD treatment.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease

Nature genetics

2022 Oct 27

Xu, Y;Kuppe, C;Perales-Patón, J;Hayat, S;Kranz, J;Abdallah, AT;Nagai, J;Li, Z;Peisker, F;Saritas, T;Halder, M;Menzel, S;Hoeft, K;Kenter, A;Kim, H;van Roeyen, CRC;Lehrke, M;Moellmann, J;Speer, T;Buhl, EM;Hoogenboezem, R;Boor, P;Jansen, J;Knopp, C;Kurth, I;Smeets, B;Bindels, E;Reinders, MEJ;Baan, C;Gribnau, J;Hoorn, EJ;Steffens, J;Huber, TB;Costa, I;Floege, J;Schneider, RK;Saez-Rodriguez, J;Freedman, BS;Kramann, R;
PMID: 36303074 | DOI: 10.1038/s41588-022-01202-z

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping

Cell reports methods

2022 Oct 24

Ali Marandi Ghoddousi, R;Magalong, VM;Kamitakahara, AK;Levitt, P;
PMID: 36313803 | DOI: 10.1016/j.crmeth.2022.100316

Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

Pages

	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

Search form

Probes for KIT

ACD’s data images for KIT gene.

Content for comparison

Gene

Product

Research area

Category

Pages

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China