Single-cell transcriptomics reveals long noncoding RNAs associated with tumor biology and the microenvironment in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is highly heterogeneous and lethal. Long noncoding RNAs (lncRNAs) are an important class of genes regulating tumorigenesis and progression. Prior bulk transcriptomic studies in PDAC have revealed the dysregulation of lncRNAs but lack single-cell resolution to distinguish lncRNAs in tumor-intrinsic biology and the tumor microenvironment (TME). We analyzed single-cell transcriptome data from 73 multiregion samples in 21 PDAC patients to evaluate lncRNAs associated with intratumoral heterogeneity and the TME in PDAC. We found 111 cell-specific lncRNAs that reflected tumor, immune and stromal cell contributions, associated with outcomes, and validated across orthogonal datasets. Single-cell analysis of tumor cells revealed lncRNAs associated with TP53 mutations and FOLFIRINOX treatment that were obscured in bulk tumor analysis. Lastly, tumor subcluster analysis revealed widespread intratumor heterogeneity and intratumoral lncRNAs associated with cancer hallmarks and tumor processes such as angiogenesis, epithelial-mesenchymal transition, metabolism and immune signaling. Intratumoral subclusters and lncRNAs were validated across six datasets and showed clinically relevant associations with patient outcomes. Our study provides the first comprehensive assessment of the lncRNA landscape in PDAC using single-cell transcriptomic data and can serve as a resource, PDACLncDB (accessible at https://www.maherlab.com/pdaclncdb-overview), to guide future functional studies.

Sonochemical Synthesis and Ion Transport Properties of Surfactant-Stabilized Carbon Nanotube Porins.

Carbon nanotube porins (CNTPs), short segments of carbon nanotubes stabilized by a lipid coating, are a promising example of artificial membrane channels that mimic a number of key behaviors of biological ion channels. While the lipid-assisted synthesis of CNTPs may facilitate their subsequent incorporation into lipid bilayers, it limits the applicability of these pores in other self-assembled membrane materials and also precludes the use of large-scale purified CNT feedstocks. Here we demonstrate that CNTPs can be synthesized by sonochemical cutting of long CNT feedstocks in the presence of different surfactants, producing CNTS with transport properties identical with those obtained by the lipid-assisted procedure. Our results open up a wide variety of synthetic routes for CNTP production.

Characterization of cell-type specific circular RNAs associated with colorectal cancer metastasis.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy and a leading cause of cancer deaths in the United States. More than half of CRC patients develop metastatic disease (mCRC) with an average 5-year survival rate of 13%. Circular RNAs (circRNAs) have recently emerged as important tumorigenesis regulators; however, their role in mCRC progression remains poorly characterized. Further, little is known about their cell-type specificity to elucidate their functions in the tumor microenvironment (TME). To address this, we performed total RNA sequencing (RNA-seq) on 30 matched normal, primary and metastatic samples from 14 mCRC patients. Additionally, five CRC cell lines were sequenced to construct a circRNA catalog in CRC. We detected 47 869 circRNAs, with 51% previously unannotated in CRC and 14% novel candidates when compared to existing circRNA databases. We identified 362 circRNAs differentially expressed in primary and/or metastatic tissues, termed circular RNAs associated with metastasis (CRAMS). We performed cell-type deconvolution using published single-cell RNA-seq datasets and applied a non-negative least squares statistical model to estimate cell-type specific circRNA expression. This predicted 667 circRNAs as exclusively expressed in a single cell type. Collectively, this serves as a valuable resource, TMECircDB (accessible at https://www.maherlab.com/tmecircdb-overview), for functional characterization of circRNAs in mCRC, specifically in the TME.

Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy.

Neoantigens are peptides derived from non-synonymous mutations presented by human leukocyte antigens (HLAs), which are recognized by antitumour T cells1-14. The large HLA allele diversity and limiting clinical samples have restricted the study of the landscape of neoantigen-targeted T cell responses in patients over their treatment course. Here we applied recently developed technologies15-17 to capture neoantigen-specific T cells from blood and tumours from patients with metastatic melanoma with or without response to anti-programmed death receptor 1 (PD-1) immunotherapy. We generated personalized libraries of neoantigen-HLA capture reagents to single-cell isolate the T cells and clone their T cell receptors (neoTCRs). Multiple T cells with different neoTCR sequences (T cell clonotypes) recognized a limited number of mutations in samples from seven patients with long-lasting clinical responses. These neoTCR clonotypes were recurrently detected over time in the blood and tumour. Samples from four patients with no response to anti-PD-1 also demonstrated neoantigen-specific T cell responses in the blood and tumour to a restricted number of mutations with lower TCR polyclonality and were not recurrently detected in sequential samples. Reconstitution of the neoTCRs in donor T cells using non-viral CRISPR-Cas9 gene editing demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Thus, effective anti-PD-1 immunotherapy is associated with the presence of polyclonal CD8+ T cells in the tumour and blood specific for a limited number of immunodominant mutations, which are recurrently recognized over time.

Breakdown of the Nernst-Einstein relation in carbon nanotube porins.

For over 100 years, the Nernst-Einstein relation has linked a charged particle's electrophoretic mobility and diffusion coefficient. Here we report experimental measurements of diffusion and electromigration of K+ ions in narrow 0.8-nm-diameter single-walled carbon nanotube porins (CNTPs) and demonstrate that the Nernst-Einstein relation in these channels breaks down by more than three orders of magnitude. Molecular dynamics simulations using polarizable force fields show that K+ ion diffusion in CNTPs in the presence of a single-file water chain is three orders of magnitude slower than bulk diffusion. Intriguingly, the simulations also reveal a disintegration of the water chain upon application of electric fields, resulting in the formation of distinct K+-water clusters, which then traverse the CNTP at high velocity. Finally, we show that although individual ion-water clusters still obey the Nernst-Einstein relation, the overall relation breaks down because of two distinct mechanisms for ion diffusion and electromigration.

RhoA with Associated TRAb or FT3 in the Diagnosis and Prediction of Graves' Ophthalmopathy.

During Graves' disease (GD) treatment, Graves' ophthalmopathy (GO) is often ignored because only mild ocular symptoms are present in early GD. Therefore, we performed isobaric tags for relative and absolute quantification (iTRAQ) analysis and measured relevant endocrine hormones to identify predisposing factors of GO. Serum samples from 3 patients with mild GD and GO and 3 patients with GD but without GO were analyzed by iTRAQ. Based on their clinical data, 60 patients with GD were divided into the GO-free and GO groups. All patients were followed up for 7 months. Their eye conditions and changes in related biochemical indexes were recorded. The iTRAQ results showed that RhoA expression was upregulated and correlated significantly with the tight junction pathway and immunity. The changes in FT3 and RhoA from baseline to 7 months, the FT3 and RhoA baseline levels, and the TRAb titer levels in patients with GD significantly differed between the groups. ELISA and western blotting for RhoA, TRAb, and FT3 in the serum samples from GO patients showed significant upregulation, as well as elevated serum RhoA and TRAb levels in the mild stage of GO. At 7 months, the serum RhoA and FT3 levels were elevated. RhoA is a potential biomarker for mild GO. In GD patients, if an elevated serum RhoA level is accompanied by an elevated TRAb or FT3 level, GO is highly likely to occur, even when obvious ocular symptoms are absent.

Sclerostin Suppression Facilitates Uveal Melanoma Progression Through Activating Wnt/ß-Catenin Signaling Via Binding to Membrane Receptors LRP5/LRP6.

Objective: Uveal melanoma (UM) is the most frequent primary eye cancer in adults with a 50% mortality rate. Characterizing the fundamental signaling pathways that drive UM is of importance for the development of targeted therapy. This study aims to probe the impact of sclerostin (SOST) on malignant progression of UM and regulation of Wnt/ß-catenin signaling. Methods: Epithelial-type (n=20) and spindle-type (n=16) UM tissues were collected for immunohistochemical staining of SOST, Wnt-1, and ß-catenin expressions. SOST was silenced in three UM cell lines (primary spindle-type OCM-1 cells, metastatic epithelial Mum-2B cells, and metastatic spindle-type Mum-2C cells) through transfecting specific siRNA. RT-qPCR and Western blot were presented for examining the levels of SOST, and markers in Wnt/ß-catenin signaling. Flow cytometry, MTT, EdU, transwell, and tube formation assays were conducted, respectively. By implanting BALB/c nude murine models in situ, the function of SOST on tumor growth was investigated, followed by immunofluorescence double staining of SOST and LRP5/6. Results: Low SOST expression as well as high Wnt-1 and ß-catenin expressions were found in epithelial-type (high malignancy) than spindle-type (low malignancy) UM tissues. Silencing SOST activated the markers in Wnt/ß-catenin signaling as well as accelerated cell cycle progression, migration, invasion, angiogenesis, and reduced apoptosis in UM cells. In situ tumor formation in murine eyes showed that SOST knockdown promoted tumor growth. Moreover, SOST interacted with LRP5/LRP6. Conclusion: SOST silencing may facilitate the malignant progression of UM cells through activating Wnt/ß-catenin signaling. Mechanistically, SOST may exert this function by interacting with LRP5/LRP6 membrane receptors.

Pandemic-Triggered Adoption of Telehealth in Underserved Communities: Descriptive Study of Pre- and Postshutdown Trends.

BACKGROUND: The adoption of telehealth services has been a challenge in rural communities. The reasons for the slow adoption of such technology-driven services have been attributed to social norms, health care policies, and a lack of infrastructure to support the delivery of services. However, the COVID-19 pandemic-related shutdown of in-person health care services resulted in the usage of telehealth services as a necessity rather than a choice. The pandemic also fast-tracked some needed legislation to allow medical cost reimbursement for remote examination and health care services. As services return to normalcy, it is important to examine whether the usage of telehealth services during the period of a shutdown has changed any of the trends in the acceptance of telehealth as a reliable alternative to traditional in-person health care services. OBJECTIVE: Our aim was to explore whether the temporary shift to telehealth services has changed the attitudes toward the usage of technology-enabled health services in rural communities. METHODS: We examined the Medicaid reimbursement data for the state of Alabama from March 2019 through June 2021. Selecting the telehealth service codes, we explored the adoption rates in 3 phases of the COVID-19 shutdown: prepandemic, pandemic before the rollout of mass vaccination, and pandemic after the rollout of mass vaccination. RESULTS: The trend in telemedicine claims had an opposite pattern to that in nontelemedicine claims across the 3 periods. The distribution of various characteristics of patients who used telemedicine (age group, gender, race, level of rurality, and service provider type) was different across the 3 periods. Claims related to behavior and mental health had the highest rates of telemedicine usage after the onset of the pandemic. The rate of telemedicine usage remained at a high level after the rollout of mass vaccination. CONCLUSIONS: The current trends indicate that adoption of telehealth services is likely to increase postpandemic and that the consumers (patients), service providers, health care establishments, insurance companies, and state and local policies have changed their attitudes toward telehealth. An increase in the use of telehealth could help local and federal governments address the shortage of health care facilities and service providers in underserved communities, and patients can get the much-needed care in a timely and effective manner.

P38 Mitogen-Activated Protein Kinase Protects Against Retinoblastoma Through Regulating USP22/SIRT1/SOST Axis.

Retinoblastoma (RB) is the most common intraocular malignancy in children. It has been previously reported that p38 MAPK is related to the pathogenesis of RB. Here we aim at investigating how p38 MAPK affected RB progression through mediating USP22/SIRT1/SOST axis. In this study, Thirty-two cases of RB and normal retinal tissues were collected. The expression of p38 MAPK, phosphorylation of p38 MAPK (P-p38 MAPK), USP22, SIRT1 and SOST in clinical tissues and cells was measured using RT-qPCR, IHC assay or western blot analysis. Cell proliferation was detected by CCK-8. Apoptosis rate of cells was examined by flow cytometry. Cell migration was evaluated using scratch test. Cell invasion ability was examined by Transwell assay. Co-immunoprecipitation (CO-IP) was utilized to measure the deubiquitination of USP22 on SIRT1. In vivo, mice were respectively injected with plasmids and the tumor growth as well as the tumor weight were detected. Results showed that p38 MAPK, P-p38 MAPK and SOST were poorly expressed in RB tissues and cells whereas USP22 and SIRT1 were overly expressed. P-p38 MAPK inhibited the expression of USP22, and overexpression of USP22 eliminated the inhibitory roles of P-p38 MAPK on tumor growth, as well as cell proliferation, migration and invasion. USP22 stabilized and promoted the expression of SIRT1 through its deubiquitination function. Silencing the expression of SIRT1 contributed to boosted expression of SOST, thus suppressing the growth of tumor cells. Collectively, the phosphorylation of p38 MAPK regulates the SIRT1/SOST axis to protect against RB via silencing USP22. The findings present some cues for a further approach to RB.

Pan-cancer proteogenomic analysis reveals long and circular noncoding RNAs encoding peptides.

Recent studies show that annotated long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) encode for stable, functional peptides that contribute to human development and disease. To systematically discover lncRNAs and circRNAs encoding peptides, we performed a comprehensive integrative analysis of mass spectrometry-based proteomic and transcriptomic sequencing data from >900 patients across nine cancer types. This enabled us to identify 19,871 novel peptides derived from 8,903 lncRNAs. Further, we exploited open reading frames overlapping the backspliced region of circRNAs to identify 3,238 peptides that are uniquely derived from 2,834 circRNAs and not their corresponding linear RNAs. Collectively, our pan-cancer proteogenomic analysis will serve as a resource for evaluating the coding potential of lncRNAs and circRNAs that could aid future mechanistic studies exploring their function in cancer.

pVACtools: A Computational Toolkit to Identify and Visualize Cancer Neoantigens.

Identification of neoantigens is a critical step in predicting response to checkpoint blockade therapy and design of personalized cancer vaccines. This is a cross-disciplinary challenge, involving genomics, proteomics, immunology, and computational approaches. We have built a computational framework called pVACtools that, when paired with a well-established genomics pipeline, produces an end-to-end solution for neoantigen characterization. pVACtools supports identification of altered peptides from different mechanisms, including point mutations, in-frame and frameshift insertions and deletions, and gene fusions. Prediction of peptide:MHC binding is accomplished by supporting an ensemble of MHC Class I and II binding algorithms within a framework designed to facilitate the incorporation of additional algorithms. Prioritization of predicted peptides occurs by integrating diverse data, including mutant allele expression, peptide binding affinities, and determination whether a mutation is clonal or subclonal. Interactive visualization via a Web interface allows clinical users to efficiently generate, review, and interpret results, selecting candidate peptides for individual patient vaccine designs. Additional modules support design choices needed for competing vaccine delivery approaches. One such module optimizes peptide ordering to minimize junctional epitopes in DNA vector vaccines. Downstream analysis commands for synthetic long peptide vaccines are available to assess candidates for factors that influence peptide synthesis. All of the aforementioned steps are executed via a modular workflow consisting of tools for neoantigen prediction from somatic alterations (pVACseq and pVACfuse), prioritization, and selection using a graphical Web-based interface (pVACviz), and design of DNA vector-based vaccines (pVACvector) and synthetic long peptide vaccines. pVACtools is available at http://www.pvactools.org.