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Open Access Publications from the University of California

Scholarly Works (8 results)

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UC San Diego Electronic Theses and Dissertations (2023)

Human cerebral cortex is tremendously complex with distinct functional areas. These functionally discrete areas lie on a large cortical sheet that has diverse cell types, cytoarchitectures, and transcriptomic variations. Previously, lots of efforts were spentto deciphering the areal cellular organizations and their molecular signatures. Recent studies that use single-cell/nucleus RNA-sequencing to profile the whole transcriptomes of human cortical areas extensively categorize the distinct transcriptomic cell types in these areas but the spatial information was lost during cell/nucleus isolation. Thus, a spatial transcriptomic tool is required to further uncover the cytoarchitectures and their spatial transcriptomes.

Recently, many spatial transcriptomic tools have emerged but have different aspects of disadvantages such as low sensitivity, low spatial resolution, and low throughput. These limitations restrict their direct application to postmortem human braintissue that have large size, general low tissue quality, and high autofluorescence signals. Here, we developed Decoded Amplified taRgeted Transcripts with Fluorescent in situ Hybridization or DART-FISH, a versatile padlock probe-based technology capable of profiling hundreds of genes in centimeter-sized human tissue sections at cellular resolution. As proof of principle, we measured 121 genes in human primary motor cortex (M1C), benchmarked, and validated its reproducibility, sensitivity, and specificity.

In addition, we applied DART-FISH to measure spatial expression of 484 genes in 9 human cortical areas along the rostrocaudal axis. Meanwhile, SNARE-seq2 was used to profile the whole transcriptomes of isolated nuclei from these areas. With the alignment of DART-FISH cells with pre-annotated SNARE-seq2 nuclei, spatially resolved transcriptomic cell atlases of human cerebral cortex were generated. In line with previous studies in M1C, these cortical areas are all composed of 24 cell subclasses of excitatory neurons, inhibitory neurons, and non-neuronal cells. Also, the found the cytoarchitectures of these cortical areas are similar except the rostral most anterior cingulate cortex (ACC) and the caudal most primary visual cortex (V1C). V1C showed the most drastic specialization, with tremendous expansion of layer 4 and increase of L4 IT neurons. To sum up, our findings suggest that these cortical areas have subtle cytoarchitectural and transcriptomic variations, except those areas at the poles of the rostrocaudal axis.

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  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

The survival rate of patients with ovarian cancer is over 90% in Stages I and II but detection strategies based on CA125 testing are ineffective. Hence, this screening approach not only fails to decrease ovarian cancer mortality but also causes significant harm including major surgery in cancer free women. Fortunately, the metrics of ultrasound-based techniques are encouraging—a recent large scale trial of over 200,000 women indicated that the sensitivity and specificity were 89.4% and 99.8%, respectively, when combined with CA125 tests. However, the positive predictive value of ovarian cancer was only 43.3%. Therefore, we hypothesize that using photoacoustic ultrasound

combined with lipid-polymer hybrid nanoparticles can achieve advances in specificity and sensitivity that are need in population-wide studies. In our study, we used poly-lacticco- glutamic acid (PLGA), lecithin, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)]-5000 (DSPE-PEG-folate) to fabricate hybrid nanoparticles. The photoacoustic imaging agents DiR or IR-1061 were encapsulated in PLGA hydrophobic core. The folate-functionalized nanoparticles can actively target HeLa cells which overexpress folate receptors on their cell membrane. The main hypothesis is that the folate-functionalized nanoparticles could produce higher

contrast between ovarian cancer tissues and normal tissues than nanoparticles without folate modification. Also, we proved our IR-1061-loaded nanoparticles had a pH-responsive release profile. We can even use photoacoustic imaging to track the releasing

IR-1061 in PBS at different pH values. In the future, we attempt to conjugate IR-1061 molecules with anti-cancer drugs to endow our nanoparticles theranostic capability. Our long-term goal is to achieve in vivo photoacoustic imaging and theranostic treatment of ovarian cancer patients.

    Cover page: Lipid-polymer hybrid nanoparticles: A photoacoustic imaging tool for ovarian cancer diagnosis
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2020)
      Cover page: Reply to Verwilt et al.: Experimental evidence against DNA contamination in SILVER-seq
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2019)
      Extracellular RNAs (exRNAs) are present in human serum. It remains unclear to what extent these circulating exRNAs may reflect human physiologic and disease states. Here, we developed SILVER-seq (Small Input Liquid Volume Extracellular RNA Sequencing) to efficiently sequence both integral and fragmented exRNAs from a small droplet (5 μL to 7 μL) of liquid biopsy. We calibrated SILVER-seq in reference to other RNA sequencing methods based on milliliters of input serum and quantified droplet-to-droplet and donor-to-donor variations. We carried out SILVER-seq on more than 150 serum droplets from male and female donors ranging from 18 y to 48 y of age. SILVER-seq detected exRNAs from more than a quarter of the human genes, including small RNAs and fragments of mRNAs and long noncoding RNAs (lncRNAs). The detected exRNAs included those derived from genes with tissue (e.g., brain)-specific expression. The exRNA expression levels separated the male and female samples and were correlated with chronological age. Noncancer and breast cancer donors exhibited pronounced differences, whereas donors with or without cancer recurrence exhibited moderate differences in exRNA expression patterns. Even without using differentially expressed exRNAs as features, nearly all cancer and noncancer samples and a large portion of the recurrence and nonrecurrence samples could be correctly classified by exRNA expression values. These data suggest the potential of using exRNAs in a single droplet of serum for liquid biopsy-based diagnostics.
        Cover page: Extracellular RNA in a single droplet of human serum reflects physiologic and disease states
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        • Peer Reviewed
        UC San Diego Previously Published Works (2021)
        We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated in vivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.
          Cover page: Revealing protein-protein interactions at the transcriptome scale by sequencing
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          • Peer Reviewed
          UC San Diego Previously Published Works (2020)
          Chromatin-associated RNA (caRNA) has been proposed as a type of epigenomic modifier. Here, we test whether environmental stress can induce cellular dysfunction through modulating RNA-chromatin interactions. We induce endothelial cell (EC) dysfunction with high glucose and TNFα (H + T), that mimic the common stress in diabetes mellitus. We characterize the H + T-induced changes in gene expression by single cell (sc)RNA-seq, DNA interactions by Hi-C, and RNA-chromatin interactions by iMARGI. H + T induce inter-chromosomal RNA-chromatin interactions, particularly among the super enhancers. To test the causal relationship between H + T-induced RNA-chromatin interactions and the expression of EC dysfunction-related genes, we suppress the LINC00607 RNA. This suppression attenuates the expression of SERPINE1, a critical pro-inflammatory and pro-fibrotic gene. Furthermore, the changes of the co-expression gene network between diabetic and healthy donor-derived ECs corroborate the H + T-induced RNA-chromatin interactions. Taken together, caRNA-mediated dysregulation of gene expression modulates EC dysfunction, a crucial mechanism underlying numerous diseases.
            Cover page: Stress-induced RNA–chromatin interactions promote endothelial dysfunction
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2023)
            The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells. These maps reveal the chromosomal domains demarcated by locally transcribed RNA, hereafter termed RNA-defined chromosomal domains. Further, the spreading of caRNA is constrained by the boundaries of topologically associating domains (TADs), demonstrating the role of the 3D genome structure in modulating the spatial distribution of RNA. Conversely, stopping transcription or acute depletion of RNA induces thousands of chromatin loops genome-wide. Activation or suppression of the transcription of specific genes suppresses or creates chromatin loops straddling these genes. Deletion of a specific caRNA-producing genomic sequence promotes chromatin loops that straddle the interchromosomal target sequences of this caRNA. These data suggest a feedback loop where the 3D genome modulates the spatial distribution of RNA, which in turn affects the dynamic 3D genome organization.
              Cover page: Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells.
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2024)
              In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.
                Cover page: Mapping human tissues with highly multiplexed RNA in situ hybridization.
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