IFNγ-Dependent Tissue-Immune Homeostasis Is Co-opted in the Tumor Microenvironment.

Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.

Alternative polyadenylation determines the functional landscape of inverted Alu repeats.

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

The SARS-CoV-2 RNA interactome.

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing.

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.

Noncanonical immune response to the inhibition of DNA methylation by Staufen1 via stabilization of endogenous retrovirus RNAs.

DNA-methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used clinically to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERVs), which can induce immune response by acting as cellular double-stranded RNAs (dsRNAs). Yet, the posttranscriptional regulation of ERV dsRNAs remains uninvestigated. Here, we find that the viral mimicry and subsequent cell death in response to decitabine require the dsRNA-binding protein Staufen1 (Stau1). We show that Stau1 directly binds to ERV RNAs and stabilizes them in a genome-wide manner. Furthermore, Stau1-mediated stabilization requires a long noncoding RNA TINCR, which enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that MDS and AML patients with lower Stau1 and TINCR expressions exhibit inferior treatment outcomes to DNMTi therapy. Overall, our study reveals the posttranscriptional regulatory mechanism of ERVs and identifies the Stau1-TINCR complex as a potential target for predicting the efficacy of DNMTis and other drugs that rely on dsRNAs.

Interpretation of an individual functional genomics experiment guided by massive public data.

A key unmet challenge in interpreting omics experiments is inferring biological meaning in the context of public functional genomics data. We developed a computational framework, Your Evidence Tailored Integration (YETI; http://yeti.princeton.edu/ ), which creates specialized functional interaction maps from large public datasets relevant to an individual omics experiment. Using this tailored integration, we predicted and experimentally confirmed an unexpected divergence in viral replication after seasonal or pandemic human influenza virus infection.

MS1-Level Proteome Quantification Platform Allowing Maximally Increased Multiplexity for SILAC and In Vitro Chemical Labeling.

Quantitative proteomic platforms based on precursor intensity in mass spectrometry (MS1-level) uniquely support in vivo metabolic labeling with superior quantification accuracy but suffer from limited multiplexity (≤3-plex) and frequent missing quantities. Here we present a new MS1-level quantification platform that allows maximal multiplexing with high quantification accuracy and precision for the given labeling scheme. The platform currently comprises 6-plex in vivo SILAC or in vitro diethylation labeling with a dedicated algorithm and is also expandable to higher multiplexity (e.g., nine-plex for SILAC). For complex samples with broad dynamic ranges such as total cell lysates, our platform performs highly accurately and free of missing quantities. Furthermore, we successfully applied our method to measure protein synthesis rate under heat shock response in human cells by 6-plex pulsed SILAC experiments, demonstrating the unique biological merits of our in vivo platform to disclose translational regulations for cellular response to stress.

The insight of in vitro and in silico studies on cholinesterase inhibitors from the roots of Cimicifuga dahurica (Turcz.) Maxim.

Cholinesterases (ChEs) are enzymes that break down neurotransmitters associated with cognitive function and memory. We isolated cinnamic acids (1 and 2), indolinones (3 and 4), and cycloartane triterpenoid derivatives (5-19) from the roots of Cimicifuga dahurica (Turcz.) Maxim. by chromatography. These compounds were evaluated for their inhibitory activity toward ChEs. Compound 1 was determined to have an IC50 value of 16.7 ± 1.9 µM, and to act as a competitive inhibitor of acetylcholinesterase (AChE). Compounds 3, 4 and 14 were found to be noncompetitive with IC50 values of 13.8 ± 1.5 and 6.5 ± 2.5 µM, and competitive with an IC50 value of 22.6 ± 0.4 µM, respectively, against butyrylcholinesterase (BuChE). Our molecular simulation suggested each key amino acid, Tyr337 of AChE and Asn228 of BuChE, which were corresponded with potential inhibitors 1, and 3 and 4, respectively. Compounds 1 and 4 were revealed to be promising compounds for inhibition of AChEs and BuChEs, respectively.

Defining cell-type specificity at the transcriptional level in human disease.

Cell-lineage-specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage-specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage-specific expression, even in lineages not separable by experimental microdissection. Our machine-learning-based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage-specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

Tissue-aware data integration approach for the inference of pathway interactions in metazoan organisms.

MOTIVATION: Leveraging the large compendium of genomic data to predict biomedical pathways and specific mechanisms of protein interactions genome-wide in metazoan organisms has been challenging. In contrast to unicellular organisms, biological and technical variation originating from diverse tissues and cell-lineages is often the largest source of variation in metazoan data compendia. Therefore, a new computational strategy accounting for the tissue heterogeneity in the functional genomic data is needed to accurately translate the vast amount of human genomic data into specific interaction-level hypotheses. RESULTS: We developed an integrated, scalable strategy for inferring multiple human gene interaction types that takes advantage of data from diverse tissue and cell-lineage origins. Our approach specifically predicts both the presence of a functional association and also the most likely interaction type among human genes or its protein products on a whole-genome scale. We demonstrate that directly incorporating tissue contextual information improves the accuracy of our predictions, and further, that such genome-wide results can be used to significantly refine regulatory interactions from primary experimental datasets (e.g. ChIP-Seq, mass spectrometry). AVAILABILITY AND IMPLEMENTATION: An interactive website hosting all of our interaction predictions is publically available at http://pathwaynet.princeton.edu. Software was implemented using the open-source Sleipnir library, which is available for download at https://bitbucket.org/libsleipnir/libsleipnir.bitbucket.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Study on the Defensive Stance and Position of Handball Goalkeepers: Facing a Forward Jump Shot Made from 9 Meters.

The purpose of this study was to find the defensive stance and calculate an optimal defense position for goalkeepers while blocking forward jump shots made from a distance of 9 m. Nine men's handball matches were recorded and 78 video clips were selected for analysis. These are the top class goalkeepers, which included players from the national team and reserve team of Korea. The goalkeeper's actual defensive position was significantly different from instructional suggestions; the width of both feet of the goalkeeper was approximately 2.5 times the width of the shoulders, and the hands were at waist height. The goalkeeper's actual defense position was about 1.10 (± 0.3) m from the goal line and also significantly different than instructional material (0.75 m). The optimal defense position, which was calculated from the goalkeeper's actual movement, was 1.44 m from the goal line, because the ratio of goalkeeper's defensive area in relation to the total area to be defended is highest at this point. In summary, we recommended that handball goalkeepers move forward, about a half step (0.34 m), when defending a forward jump shot made from 9 m, and instructional material should be modified according to the findings from this study.

Ontology-aware classification of tissue and cell-type signals in gene expression profiles across platforms and technologies.

MOTIVATION: Leveraging gene expression data through large-scale integrative analyses for multicellular organisms is challenging because most samples are not fully annotated to their tissue/cell-type of origin. A computational method to classify samples using their entire gene expression profiles is needed. Such a method must be applicable across thousands of independent studies, hundreds of gene expression technologies and hundreds of diverse human tissues and cell-types. RESULTS: We present Unveiling RNA Sample Annotation (URSA) that leverages the complex tissue/cell-type relationships and simultaneously estimates the probabilities associated with hundreds of tissues/cell-types for any given gene expression profile. URSA provides accurate and intuitive probability values for expression profiles across independent studies and outperforms other methods, irrespective of data preprocessing techniques. Moreover, without re-training, URSA can be used to classify samples from diverse microarray platforms and even from next-generation sequencing technology. Finally, we provide a molecular interpretation for the tissue and cell-type models as the biological basis for URSA's classifications.

Deadenylation kinetics of mixed poly(A) tails at single-nucleotide resolution.

Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.

Pelvic floor muscle training using an extracorporeal biofeedback device for female stress urinary incontinence.

INTRODUCTION AND HYPOTHESIS: Extracorporeal biofeedback was developed to reduce patient discomfort when performing strengthening exercises. The efficacy and safety of extracorporeal biofeedback combined with pelvic floor muscle training (PFMT) for the treatment of female stress urinary incontinence (SUI) were evaluated. METHODS: One hundred and six participants with SUI were enrolled in a 12-week PFMT program using extracorporeal biofeedback intervention. A standard pad test was performed, and pelvic floor muscle strength was assessed using the Oxford scale. Measurements were taken with a perineometer at baseline and at a 12-week follow-up visit. An objective cure was defined as less than 2 g of urine leakage by the standard pad test. The long-term effects of extracorporeal biofeedback and PFMT were investigated by interviewing the participants 12 months after treatment. RESULTS: Seventy-one participants completed the 12-week extracorporeal biofeedback intervention. The objective cure rate was 52.1 %, and there was a significant reduction in pad weight over the time period. The incontinence visual analogue scale, the Sandvik severity index, and the incontinence quality-of-life questionnaire domains were significantly improved after treatment (p<0.001). The strength of the PFM was significantly increased after the 12-week treatment. After PFMT, 64.3 % of 56 participants reported good treatment compliance, and 24 participants (42.9 %) had continued PFMT at home 12 months after treatment. Age and baseline pad weight were negative predictive factors for an objective cure of SUI. CONCLUSIONS: Pelvic floor muscle training using extracorporeal biofeedback can be an effective and safe conservative treatment option for female SUI without the discomfort caused by vaginal sensors.

Diagnostic role of prostate resection in the elderly patients who experience significant co-morbidity with a high clinical suspicion of prostate cancer.

The necessity of routine prostate biopsy prior to transurethral resection of the prostate (TURP) in elderly comorbid patients with a high prostate specific antigen (PSA) level remains controversial. We assessed the role of TURP in prostate cancer diagnosis in these individuals. A total of 197 patients underwent TURP in conjunction with prostatic needle biopsy. Pathologic reviews of specimens of TUR chips and biopsy cores were analyzed. Overall, prostate cancer (CaP) was detected in 114 patients (57.6%). Ninety-eight cancers (86%) were detected with TURP and biopsy, and seven cancers (6.1%) with only TURP. The Gleason score of a TUR-specimen was identical to that of the biopsy-core in 43.9% of cases. Variables associated with diagnostic accuracy in the TUR-specimens included the prebiopsy PSA level, prostate specific antigen density (PSAD), and the Gleason score in biopsy cores. In patients with a PSA level and a PSAD that was greater than 15.4 ng/mL and 0.69 ng/mL/g, respectively, 100% of the cancers were detected in the TUR-specimens. Our results suggest that a prostatic biopsy might be omitted prior to TURP in elderly patients with significant co-morbidity and levels for PSA of >15.4 ng/mL.

Short poly(A) tails are protected from deadenylation by the LARP1-PABP complex.

Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.

Single-center study on clinicopathological and typical molecular pathologic features of metastatic brain tumor.

BACKGROUND: The metastatic brain tumor is the most common brain tumor. The aim of this study was to demonstrate the clinicopathological and molecular pathologic features of brain metastases (BM). METHODS: A total of 269 patients were diagnosed with BM through surgical resection at Seoul St. Mary's Hospital from January 2010 to March 2020. We reviewed the clinicopathological features and molecular status of primary and metastatic brain tissues using immunohistochemistry and molecular pathology results. RESULTS: Among 269 patients, 139 males and 130 females were included. The median age of primary tumor was 58 years (range, 13 to 87 years) and 86 patients (32.0%) had BM at initial presentation. Median BM free interval was 28.0 months (range, 1 to 286 months). The most frequent primary site was lung 46.5% (125/269), and followed by breast 15.6% (42/269), colorectum 10.0% (27/269). Epidermal growth factor receptor (EGFR) mutation was found in 50.8% (32/63) and 58.0% (40/69) of lung primary and BM, respectively. In both breast primary and breast cancer with BM, luminal B was the most frequent subtype at 37.9% (11/29) and 42.9% (18/42), respectively, followed by human epidermal growth factor receptor 2 with 31.0% (9/29) and 33.3% (14/42). Triple-negative was 20.7% (6/29) and 16.7% (7/42), and luminal A was 10.3% (3/29) and 7.1% (3/42) of breast primary and BM, respectively. In colorectal primary and colorectal cancer with BM, KRAS mutation was found in 76.9% (10/13) and 66.7% (2/3), respectively. CONCLUSIONS: We report the clinicopathological and molecular pathologic features of BM that can provide useful information for understanding the pathogenesis of metastasis and for clinical trials based on the tumor's molecular pathology.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research.

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM(+)/CD44(+) population accounts for 4.5% of tumor cells. EpCAM(+)/CD44(+) gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) and EpCAM(-)/CD44(+) cells failed to do so. Xenografts of EpCAM(+)/CD44(+) gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM(+)/CD44(+), but not EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) or EpCAM(-)/CD44(+) cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM(+)/CD44(+) cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM(+)/CD44(+) subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

The m6A(m)-independent role of FTO in regulating WNT signaling pathways.

FTO and ALKBH5 are the two enzymes responsible for mRNA demethylation. Hence, the functional study of FTO has been focused on its mechanistic role in dynamic mRNA modification, and how this post-transcriptional regulation modulates signaling pathways. Here, we report that the functional landscape of FTO is largely associated with WNT signaling pathways but in a manner that is independent of its enzymatic activity. Re-analyses of public datasets identified the bifurcation of canonical and noncanonical WNT pathways as the major role of FTO. In FTO-depleted cells, we find that the canonical WNT/ß-Catenin signaling is attenuated in a non-cell autonomous manner via the up-regulation of DKK1. Simultaneously, this up-regulation of DKK1 promotes cell migration via activating the noncanonical WNT/PCP pathway. Unexpectedly, this regulation of DKK1 is independent of its RNA methylation status but operates at the transcriptional level, revealing a noncanonical function of FTO in gene regulation. In conclusion, this study places the functional context of FTO at the branch point of multiple WNT signaling pathways and extends its mechanistic role in gene regulation.