circTulp4 functions in Alzheimer\'s disease pathogenesis by regulating its parental gene, Tulp4

Molecular therapy : the journal of the American Society of Gene Therapy

2021 Feb 10

Ma, N;Pan, J;Wen, Y;Wu, Q;Yu, B;Chen, X;Wan, J;Zhang, W;
PMID: 33578037 | DOI: 10.1016/j.ymthe.2021.02.008

Alzheimer's disease (AD)-one of the most common neurodegenerative diseases worldwide-impairs cognition, memory, and language ability and causes dementia. However, AD pathogenesis remains poorly elucidated. Recently, a potential link between AD and circular RNAs (circRNAs) has been uncovered, but only a few circRNAs that might be involved in AD have been identified. Here, we systematically investigated circRNAs in the APP/PS1 model mouse brain through deep RNA sequencing. We report that circRNAs are markedly enriched in the brain and that several circRNAs exhibit differential expression between wild-type and APP/PS1 mice. We characterized one abundant circRNA, circTulp4, derived from Intron1 of the gene Tulp4. Our results indicate that circTulp4 predominantly localizes in the nucleus and interacts with U1 small nuclear ribonucleoprotein particle (snRNP) and RNA polymerase II to modulate the transcription of its parental gene, Tulp4, and thereby regulate the function of the nervous system, and might participate in the development of AD.

Mitohormesis in Hypothalamic POMC Neurons Mediates Regular Exercise-Induced High-Turnover Metabolism

Cell metabolism

2021 Feb 02

Kang, GM;Min, SH;Lee, CH;Kim, JY;Lim, HS;Choi, MJ;Jung, SB;Park, JW;Kim, S;Park, CB;Dugu, H;Choi, JH;Jang, WH;Park, SE;Cho, YM;Kim, JG;Kim, KG;Choi, CS;Kim, YB;Lee, C;Shong, M;Kim, MS;
PMID: 33535098 | DOI: 10.1016/j.cmet.2021.01.003

Low-grade mitochondrial stress can promote health and longevity, a phenomenon termed mitohormesis. Here, we demonstrate the opposing metabolic effects of low-level and high-level mitochondrial ribosomal (mitoribosomal) stress in hypothalamic proopiomelanocortin (POMC) neurons. POMC neuron-specific severe mitoribosomal stress due to Crif1 homodeficiency causes obesity in mice. By contrast, mild mitoribosomal stress caused by Crif1 heterodeficiency in POMC neurons leads to high-turnover metabolism and resistance to obesity. These metabolic benefits are mediated by enhanced thermogenesis and mitochondrial unfolded protein responses (UPRmt) in distal adipose tissues. In POMC neurons, partial Crif1 deficiency increases the expression of β-endorphin (β-END) and mitochondrial DNA-encoded peptide MOTS-c. Central administration of MOTS-c or β-END recapitulates the adipose phenotype of Crif1 heterodeficient mice, suggesting these factors as potential mediators. Consistently, regular running exercise at moderate intensity stimulates hypothalamic MOTS-c/β-END expression and induces adipose tissue UPRmt and thermogenesis. Our findings indicate that POMC neuronal mitohormesis may underlie exercise-induced high-turnover metabolism.

Inhibition of GABAA-ρ receptors induces retina regeneration in zebrafish

Neural Regen Res

2021 Feb 01

Kent, MR;Kara, N;Patton, JG;
PMID: 32859800 | DOI: 10.4103/1673-5374.286972

A potential treatment for retinal diseases is to induce an endogenous Müller glia (MG)-derived regenerative response to replace damaged neurons. In contrast to mammalian MG, zebrafish MG are capable of mediating spontaneous regeneration. We seek to define the mechanisms that enable retina regeneration in zebrafish in order to identify therapeutic targets to induce mammalian retina regeneration. We previously used pharmacological and genetic methods to inhibit gamma aminobutyric acid A (GABAA) receptors in undamaged zebrafish retinas and showed that such inhibition could induce initiation of retina regeneration, as measured by the dedifferentiation of MG and the appearance of MG-derived proliferating progenitor cells. Here, we show that inhibition of a pharmacologically distinct subset of GABAA receptors (GABAA-ρ) can also induce retina regeneration. Dual inhibition of both GABA receptor subtypes led to enhanced retina regeneration. Gene expression analyses indicate that inhibition of GABAA-ρ receptors induces a canonical retinal regenerative response. Our results support a model in which decreased levels of GABA, such as would occur after retinal cell death or damage, induce dedifferentiation of MG and the generation of proliferating progenitor cells during zebrafish retina regeneration. Animal experiments were approved by the Vanderbilt's Institutional Animal Care and Use Committee (Protocol M1800200) on January 29, 2019.

Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses

Nature microbiology

2021 Jan 28

Chen, W;Foo, SS;Hong, E;Wu, C;Lee, WS;Lee, SA;Evseenko, D;Moreira, MEL;García-Sastre, A;Cheng, G;Nielsen-Saines, K;Brasil, P;Avvad-Portari, E;Jung, JU;
PMID: 33510473 | DOI: 10.1038/s41564-020-00850-3

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV+ pericytes of fetal human brain specimens and in vertically transmitted ZIKV+ human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.

Derivation of snake venom gland organoids for in vitro venom production

Nature protocols

2021 Jan 27

Puschhof, J;Post, Y;Beumer, J;Kerkkamp, HM;Bittenbinder, M;Vonk, FJ;Casewell, NR;Richardson, MK;Clevers, H;
PMID: 33504990 | DOI: 10.1038/s41596-020-00463-4

More than 400,000 people each year suffer adverse effects following bites from venomous snakes. However, snake venom is also a rich source of bioactive molecules with known or potential therapeutic applications. Manually 'milking' snakes is the most common method to obtain venom. Safer alternative methods to produce venom would facilitate the production of both antivenom and novel therapeutics. This protocol describes the generation, maintenance and selected applications of snake venom gland organoids. Snake venom gland organoids are 3D culture models that can be derived within days from embryonic or adult venom gland tissues from several snake species and can be maintained long-term (we have cultured some organoids for more than 2 years). We have successfully used the protocol with glands from late-stage embryos and recently deceased adult snakes. The cellular heterogeneity of the venom gland is maintained in the organoids, and cell type composition can be controlled through changes in media composition. We describe in detail how to derive and grow the organoids, how to dissociate them into single cells, and how to cryopreserve and differentiate them into toxin-producing organoids. We also provide guidance on useful downstream assays, specifically quantitative real-time PCR, bulk and single-cell RNA sequencing, immunofluorescence, immunohistochemistry, fluorescence in situ hybridization, scanning and transmission electron microscopy and genetic engineering. This stepwise protocol can be performed in any laboratory with tissue culture equipment and enables studies of venom production, differentiation and cellular heterogeneity.

Low-Dose Ad26.COV2.S Protection Against SARS-CoV-2 Challenge in Rhesus Macaques

bioRxiv : the preprint server for biology

2021 Jan 27

He, X;Chandrashekar, A;Zahn, R;Wegmann, F;Yu, J;Mercado, NB;McMahan, K;Martinot, AJ;Piedra-Mora, C;Beecy, S;Ducat, S;Chamanza, R;Huber, SR;van der Fits, L;Borducchi, EN;Lifton, M;Liu, J;Nampanya, F;Patel, S;Peter, L;Tostanoski, LH;Pessaint, L;Van Ry, A;Finneyfrock, B;Velasco, J;Teow, E;Brown, R;Cook, A;Andersen, H;Lewis, MG;Schuitemaker, H;Barouch, DH;
PMID: 33532782 | DOI: 10.1101/2021.01.27.428380

We previously reported that a single immunization with an adenovirus serotype 26 (Ad26) vector-based vaccine expressing an optimized SARS-CoV-2 spike (Ad26.COV2.S) protected rhesus macaques against SARS-CoV-2 challenge. In this study, we evaluated the immunogenicity and protective efficacy of reduced doses of Ad26.COV2.S. 30 rhesus macaques were immunized once with 1×10 11 , 5×10 10 , 1.125×10 10 , or 2×10 9 vp Ad26.COV2.S or sham and were challenged with SARS-CoV-2 by the intranasal and intratracheal routes. Vaccine doses as low as 2×10 9 vp provided robust protection in bronchoalveolar lavage, whereas doses of 1.125×10 10 vp were required for protection in nasal swabs. Activated memory B cells as well as binding and neutralizing antibody titers following vaccination correlated with protective efficacy. At suboptimal vaccine doses, viral breakthrough was observed but did not show evidence of virologic, immunologic, histopathologic, or clinical enhancement of disease compared with sham controls. These data demonstrate that a single immunization with a relatively low dose of Ad26.COV2.S effectively protected against SARS-CoV-2 challenge in rhesus macaques. Moreover, our findings show that a higher vaccine dose may be required for protection in the upper respiratory tract compared with the lower respiratory tract.

Fatal neuroinvasion of SARS-CoV-2 in K18-hACE2 mice is partially dependent on hACE2 expression

bioRxiv : the preprint server for biology

2021 Jan 15

Carossino, M;Montanaro, P;O'Connell, A;Kenney, D;Gertje, H;Grosz, KA;Kurnick, SA;Bosmann, M;Saeed, M;Balasuriya, UBR;Douam, F;Crossland, NA;
PMID: 33469581 | DOI: 10.1101/2021.01.13.425144

Animal models recapitulating the distinctive features of severe COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. However, the cause(s) and mechanisms of lethality in this mouse model remain unclear. Here, we evaluated the spatiotemporal dynamics of SARS-CoV-2 infection for up to 14 days post-infection. Despite infection and moderate inflammation in the lungs, lethality was invariably associated with viral neuroinvasion and neuronal damage (including spinal motor neurons). Neuroinvasion occurred following virus transport through the olfactory neuroepithelium in a manner that was only partially dependent on hACE2. Interestingly, SARS-CoV-2 tropism was overall neither widespread among nor restricted to only ACE2-expressing cells. Although our work incites caution in the utility of the K18-hACE2 model to study global aspects of SARS-CoV-2 pathogenesis, it underscores this model as a unique platform for exploring the mechanisms of SARS-CoV-2 neuropathogenesis. COVID-19 is a respiratory disease caused by SARS-CoV-2, a betacoronavirus. Here, we show that in a widely used transgenic mouse model of COVID-19, lethality is invariably associated with viral neuroinvasion and the ensuing neuronal disease, while lung inflammation remains moderate.

Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics

Cell

2021 Jan 13

Gengatharan, A;Malvaut, S;Marymonchyk, A;Ghareghani, M;Snapyan, M;Fischer-Sternjak, J;Ninkovic, J;Götz, M;Saghatelyan, A;
PMID: 33482084 | DOI: 10.1016/j.cell.2020.12.026

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized in vivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. In vivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

Stem cell-derived CAR T cells traffic to HIV reservoirs in macaques

JCI insight

2021 Jan 11

Barber-Axthelm, IM;Barber-Axthelm, V;Sze, KY;Zhen, A;Suryawanshi, GW;Chen, IS;Zack, JA;Kitchen, SG;Kiem, HP;Peterson, CW;
PMID: 33427210 | DOI: 10.1172/jci.insight.141502

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

Preliminary RNA-microarray analysis of long non-coding RNA expression in abnormally invasive placenta

Exp Ther Med

2021 Jan 01

Zhang, H;Wu, S;Ye, S;Ma, H;Liu, Z;
PMID: 33235622 | DOI: 10.3892/etm.2020.9445

Long non-coding RNAs (lncRNAs) are reported to have important roles in placental development and function, but the role of lncRNAs in abnormally invasive placenta (AIP) remains elusive. In the present study, the differential expression profiles of lncRNAs were analyzed to identify novel targets for further study of AIP. A total of 10 lncRNAs were chosen for validation by reverse transcription-quantitative PCR. To further determine the functions of dysregulated lncRNAs and their corresponding mRNAs, functional enrichment analysis, coexpression analysis were performed. A total of 329 lncRNAs and 179 mRNAs were identified to be differently expressed between the invasive and control group. Gene ontology analysis revealed that the 10 most significantly enriched functions included upregulated mRNAs and the most significantly enriched term was related to the proteinaceous extracellular matrix (ECM). In the pathway analysis, the two most significantly enriched pathways were the TGF-β signaling pathway for upregulated mRNAs and the pentose phosphate pathway for downregulated mRNAs. Furthermore, for certain dysregulated lncRNAs, their associated mRNAs were also dysregulated. Of note, BMP and activin membrane-bound inhibitor and TGF-β-induced, as the target genes of the TGF-β pathway, were indicated to be closely related to the ECM and invasive placental cells. Their nearby lncRNAs G008916 and vault RNA2-1 were also significantly dysregulated. In conclusion, significant lncRNAs with the potential to serve as biomarkers for AIP were identified.

Resolution of hypereosinophilic syndrome following resection of a schwannoma

The journal of allergy and clinical immunology. In practice

2023 Jan 05

Ware, JM;Folio, LR;Pittaluga, S;Klion, A;Khoury, P;
PMID: 36621605 | DOI: 10.1016/j.jaip.2022.12.028

The technological landscape and applications of single-cell multi-omics

Nature reviews. Molecular cell biology

2023 Jun 06

Baysoy, A;Bai, Z;Satija, R;Fan, R;
PMID: 37280296 | DOI: 10.1038/s41580-023-00615-w

Single-cell multi-omics technologies and methods characterize cell states and activities by simultaneously integrating various single-modality omics methods that profile the transcriptome, genome, epigenome, epitranscriptome, proteome, metabolome and other (emerging) omics. Collectively, these methods are revolutionizing molecular cell biology research. In this comprehensive Review, we discuss established multi-omics technologies as well as cutting-edge and state-of-the-art methods in the field. We discuss how multi-omics technologies have been adapted and improved over the past decade using a framework characterized by optimization of throughput and resolution, modality integration, uniqueness and accuracy, and we also discuss multi-omics limitations. We highlight the impact that single-cell multi-omics technologies have had in cell lineage tracing, tissue-specific and cell-specific atlas production, tumour immunology and cancer genetics, and in mapping of cellular spatial information in fundamental and translational research. Finally, we discuss bioinformatics tools that have been developed to link different omics modalities and elucidate functionality through the use of better mathematical modelling and computational methods.

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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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