Tetracyclines revisited: tetracyclines in the field of dermatology.

Tetracyclines are a class of broad-spectrum antibiotics favored by dermatologists. Over the last decade, the clinical efficacy of tetracyclines has expanded into various dermatoses. This review tries to encompass the possible indications of tetracycline in the field of dermatology and possible mechanisms of action. This comprehensive review encompasses all possible indications of tetracyclines besides acne vulgaris and rosacea: hidradenitis suppurativa, autoimmune bullous dermatoses, vitiligo, alopecia, prurigo pigmentosa, granulomatous dermatoses, Kaposi sarcoma, cold urticaria, atopic dermatitis, scrub typhus, scarring, and miscellaneous dermatoses. We also focus on the recently approved sarecycline, a third generation narrow-spectrum tetracycline, and its clinical efficacy and potential impact on the microbiome. Our review provides a better understanding of this extremely familiar drug class and encourages its use in a wider spectrum of dermatologic diseases and symptoms.

Prevalence and Incidence of Psoriatic Arthritis among Patients with Psoriasis and Risk Factors for Psoriatic Arthritis in Republic of Korea: A Nationwide Database Cohort Study.

Population-based epidemiological studies on disease burden and risk factors for psoriatic arthritis (PsA) in patients with psoriasis (PsO) are limited, especially in Asian populations. Therefore, the aim was to determine the prevalence and incidence of PsA among PsO patients in Korea, and examine associated clinical factors. A cohort study was performed to determine the annual prevalence and incidence of PsA among PsO patients between 2008 and 2020 using nationwide claims data in Korea. Risk factors for PsA development were also examined using logistic regression among matched PsA cases and controls. An increasing trend in PsA prevalence per 1,000 patients was observed; prevalence was 6.17 (95% confidence interval [CI] 5.73-6.65) in 2008 and 19.03 (95% CI 18.39-19.70) in 2020. Similarly, the PsA incidence rate per 1,000 patient-years increased from 3.35 (95% CI 3.01-3.72) in 2008 to 5.01 (95% CI 4.68-5.36) in 2020. Patients with plaque PsO, moderate-to severe PsO, receiving oral systemic therapy or phototherapy, with a higher burden of comorbidities, and concomitant autoimmune diseases had a higher risk of PsA. The results provide insight into the burden of PsA among PsO patients in Korea and risk factors associated with developing PsA.

Single-cell RNA sequencing of a poorly metastatic melanoma cell line and its subclones with high lung and brain metastasis potential reveals gene expression signature of metastasis with prognostic implication.

The molecular mechanisms underlying melanoma metastasis remain poorly understood. In this study, we aimed to delineate the mechanisms underlying gene expression alterations during metastatic potential acquisition and characterize the metastatic subclones within primary cell lines. We performed single-cell RNA sequencing of a poorly metastatic melanoma cell line (WM239A) and its subclones with high metastatic potential to the lung (113/6-4L) and the brain (131/4-5B1 and 131/4-5B2). Unsupervised clustering of 8173 melanoma cells identified three distinct clusters according to cell type ('Primary', 'Lung' and 'Brain' clusters) with differential expression of MITF and AXL pathways and putative cancer and cell cycle drivers, with the lung cluster expressing intermediate but distinct gene profiles between primary and brain clusters. Principal component (PC) analysis revealed that PC2 (the second PC), which was positively associated with MITF expression and negatively with AXL pathways, primarily segregated cell types, in addition to PC1 of the cell cycle pathway. Pseudotime trajectory and RNA velocity analyses suggested the existence of cellular subsets with metastatic potential in the Primary cluster and an association between PC2 signature alteration and metastasis potential acquisition. Analysis of The Cancer Genome Atlas melanoma samples by clustering into PC2-high and -low clusters by quartiles of PC2 signature expression revealed that the PC2-high cluster was an independent significant factor for poor prognosis (p-value = 0.003) with distinct genomic and transcriptomic characteristics, compared to the PC2-low cluster. In conclusion, we identified signatures of melanoma metastasis with prognostic significance and putative pro-metastatic subclones within a primary cell line.

Targeted deep sequencing reveals genomic alterations of actinic keratosis/cutaneous squamous cell carcinoma in situ and cutaneous squamous cell carcinoma.

Actinic keratosis (AK) and cutaneous squamous cell carcinoma in situ (CIS) are two of the most common precursors of cutaneous squamous cell carcinoma (cSCC). However, the genomic landscape of AK/CIS and the drivers of cSCC progression remain to be elucidated. The aim of our study was to investigate the genomic alterations between AK/CIS and cSCC in terms of somatic mutations and copy number alterations (CNAs). We performed targeted deep sequencing of 160 cancer-related genes with a median coverage of 515× for AK (N = 9), CIS (N = 9), cSCC lesions (N = 13), and matched germline controls from 17 patients. cSCC harboured higher abundance of total mutations, driver mutations and CNAs than AK/CIS. Driver mutations were found in TP53 (81%), NOTCH1 (32%), RB1 (26%) and CDKN2A (19%). All AK/CIS and cSCC lesions (93.5%), except two, harboured TP53 or NOTCH1 mutations, some of which were known oncogenic mutations or reported mutations in normal skin. RB1 driver mutations were found in CIS/cSCC (36.4%) but not in AK. CDKN2A driver mutations were found more frequently in cSCC (30.8%) than in AK/CIS (11.1%). Among recurrent (≥3 samples) CNAs (gain in MYC and PIK3CA/SOX2/TP63; loss in CDKN2A and RB1), MYC (8q) gain and CDKN2A (9p) loss were more frequently detected in cSCC (30.8%) than in AK/CIS (11.1%). Ultraviolet was responsible for the majority of somatic mutations in both AK/CIS and cSCC. Our study revealed that AK/CIS lesions harbour prevalent TP53 or NOTCH1 mutations and that additional somatic mutations and CNAs may lead to cSCC progression in AK/CIS lesions.

Deep brain stimulation in Parkinson disease with valosin-containing protein gene mutation.

BACKGROUND AND PURPOSE: Mutations in the gene encoding valosin-containing protein (VCP) are related to myriad medical conditions, including familial amyotrophic lateral sclerosis, inclusion body myopathy, and frontotemporal dementia. There are several reports of a link between these mutations and early onset Parkinson disease (PD). CASE DESCRIPTION: We report a 53-year-old PD patient with VCP mutation who later developed motor complications, thus receiving subthalamic nucleus deep brain stimulation (DBS) at the age of 56 years. However, myopathy emerged 1.5 years after surgery. CONCLUSIONS: With the phenotype variability of VCP, DBS should be carefully evaluated, considering the possible unfavorable long-term outcomes due to other symptoms of this mutation.

Single-cell RNA sequencing reveals the existence of pro-metastatic subpopulation within a parental B16 murine melanoma cell line.

The mechanism of melanoma metastasis is poorly understood, especially at the single-cell level. To understand the evolution from primary melanoma to metastasis, we investigated single-cell transcriptome profiles of parental B16 melanoma cells (B16F0) and its highly metastatic subclone (B16F10). Genomic alterations between cells were also analyzed by whole-exome sequencing. We identified 274 differentially expressed genes (DEGs) in B16F10, including upregulated genes related to metastasis, Lgals3, Sparc, Met, and Tmsb4x, and downregulated Mitf pathway genes, Ptgds, Cyb5a, and Cd63. The proportion of cycling cells and cells highly expressing Kdm5b was significantly high in B16F10 cells. Among the five subclusters of B16 cells (C1-5), C3/C4 clusters comprised both B16F0 and B16F10 cells and exhibited intermediate DEG patterns, whereas the C5 cluster mostly comprised B16F10 and showed typical metastatic characteristics. In trajectory analysis, the C4 cluster in B16F0, which showed unique characteristics (mainly cycling cells and upregulation of Mitf pathway genes), have transition potential to the C5 cluster (B16F10). Regarding genomic alterations, stepwise evolution with shared mutations, including Braf, Pten, and Trp53, and further specific alterations led to metastatic development. Our results provide deeper understanding of melanoma metastasis at the single-cell level, thus aiding further studies in melanoma metastasis control.

Encephalitis with status epilepticus and stroke as complications of non-severe COVID-19 in a young female patient: a case report.

BACKGROUND: Neurological manifestations of COVID-19 are thought to be associated with the disease severity of COVID-19 and poor clinical outcomes. Dysregulated immune responses are considered to be mediating such complications. Our case illustrates multiple critical neurological complications simultaneously developed in a patient with non-severe COVID-19 and successful recovery with a multifaceted therapeutic approach. The cerebrospinal fluid (CSF) interleukin-6 (IL-6) level was temporally correlated with the clinical severity of the status epilepticus in our patient, suggesting a causal relationship. CASE PRESENTATION: A previously healthy 20-year-old female patient presented with a first-onset seizure. Concomitant non-severe COVID-19 pneumonia was diagnosed. CSF study showed lymphocytic pleocytosis with elevated IL-6 levels in CSF. During hospitalization under the diagnosis of autoimmune encephalitis, status epilepticus developed, and the seizure frequency was temporally correlated with the CSF IL-6 level. Furthermore, a new embolic stroke developed without a significant cardioembolic source. Contrary to the exacerbated COVID-19-associated neurological complications, COVID-19 pneumonia was cleared entirely. After treatment with antiseizure medications, antithrombotics, antiviral agents, and immunotherapy, the patient was discharged with near-complete recovery. CONCLUSION: Active serological, and radiological evaluation can be helpful even in non-severe COVID-19, and multidimensional treatment strategies, including immunotherapy, can successfully reverse the neurological complication.

Pathogenic NF1 truncating mutation and copy number alterations in a dedifferentiated liposarcoma with multiple lung metastasis: a case report.

BACKGROUND: Dedifferentiated liposarcoma (DDLPS), which accounts for an estimated 15-20% of liposarcomas, is a high-grade and aggressive malignant neoplasm, exhibiting a poor response to available therapeutic agents. However, genetic alteration profiles of DDLPS as well as the role of NF1 mutations have not been studied extensively. CASE PRESENTATION: The current study reports a patient presenting with rapidly growing DDLPS accompanied by multiple lung and pleural metastases, in whom whole-exome sequencing revealed a NF1 truncating mutation of the known pathogenic variant, c.C7486T, p.R2496X, as well as multiple copy number alterations (CNAs), including the well-known 12q13-15 amplification, and multiple chromothripsis events encompassing potential cancer-related genes. CONCLUSIONS: Our results suggest that, in addition to the 12q13-15 amplification, NF1 inactivation mutation and other CNAs may contribute to DDLPS tumorigenesis accompanied by aggressive clinical features.

Multiple Spitz nevi after allogeneic hematopoietic stem cell transplantation.

Spitz nevus commonly appears as a solitary lesion. A 12-year-old male patient presented with a 6-month history of several pigmented lesions on his trunk and lower extremities. He had undergone chemoradiotherapy and unrelated umbilical cord blood transplantation against recurring acute lymphoblastic leukemia for 6 years. After that, several pigmented lesions abruptly developed on his trunk and lower extremities, and the number of those increased significantly. Pathologically, the diagnosis of multiple Spitz nevi was made. In a clinical correlation, we diagnosed multiple Spitz nevi resulting from such an immunocompromised condition. This is the first description of clinical, dermoscopic, and histopathologic features of multiple Spitz nevi in the hematopoietic cell transplantation (HSCT) recipient child.

Mutational Landscapes of Normal Skin and Their Potential Implications in the Development of Skin Cancer: A Comprehensive Narrative Review.

Recent evidence suggests that physiologically normal skin harbors pervasive mutant clones with cancer drivers. Normal skin has the highest burden of somatic mutations due to persistent ultraviolet exposure throughout life. The mutation burden exponentially increases with age and is further modified by skin site, sun-damage history, and skin phototype. Driver gene profiles in normal skin are similar to those in cutaneous squamous cell carcinoma where NOTCH family, FAT family, and TP53 are consistently reported, while other reported profiles include PPM1D, KMT2D, ASXL1, and RBM10. Normal skin seldom harbors canonical hotspot mutations with therapeutic relevance. The pathologic role of mutant clones with cancer drivers in normal skin is classically considered precursors for skin cancer; however, recent evidence also suggests their putative cancer-protective role. Copy number alterations and other structural variants are rare in normal skin with loss in 9q region encompassing NOTCH1 being the most common. Study methodologies should be carefully designed to obtain an adequate number of cells for sequencing, and a comparable number of cells and read depth across samples. In conclusion, this review provides mutational landscapes of normal skin and discusses their potential implications in the development of skin cancer, highlighting the role of driver genes in early malignant progression.

A double CYP27A1 gene mutation in spinal cerebrotendinous xanthomatosis in a patient presenting with spastic gait: a case report.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX, OMIM #213700) is a rare inherited metabolic disease caused by the mutation in the CYP27A1 gene. Spinal CTX is a rare clinical subgroup of CTX which lacks typical symptoms seen in classical CTX. Here we report a spinal CTX case revealed double mutation of CYP27A1 gene. CASE PRESENTATION: A 42-year-old Asian man visited our hospital with spastic gait started at 35. Physical examination showed bilateral masses on his Achilles tendons and were identified as xanthoma on ankle magnetic resonance imaging (MRI). Brain and spinal cord MRI revealed high signal lesions in bilateral cerebellar dentate nuclei and long tract lesions involving lateral corticospinal and gracile tracts. Gene analysis revealed double heterozygous mutation, c.223C > T (p. Gln75Ter) and c.1214G > A (p. Arg405Gln). CONCLUSIONS: We believe that novel mutation detected in our case might have a role in the pathomechanism in CTX. Moreover, spinal CTX should be considered in the patients only presenting with pyramidal symptoms, as CTX shows good prognosis in early treatment with chenodeoxycholic acid.

Intratumoral IL-12 delivery via mesenchymal stem cells combined with PD-1 blockade leads to long-term antitumor immunity in a mouse glioblastoma model.

BACKGROUND: Although PD-1 blockade is effective for treating several types of cancer, the efficacy of this agent in glioblastoma is largely limited. To overcome non-responders and the immunosuppressive tumor microenvironment, combinational immunotherapeutic strategies with anti-PD-1 need to be considered. Here, we developed IL-12-secreting mesenchymal stem cells (MSC_IL-12) with glioblastoma tropism and evaluated the therapeutic effects of anti-PD-1, MSC_IL-12, and their combination against glioblastoma. METHODS: Therapeutic responses were evaluated using an immunocompetent mouse orthotopic model. Tumor-infiltrating lymphocytes (TILs) were analyzed using immunofluorescent imaging. Single-cell transcriptome was obtained from mouse brains after treatments. RESULTS: Anti-PD-1 and MSC_IL-12 showed complete tumor remission in 25.0% (4/16) and 23.1% (3/13) of glioblastoma-implanted mice, respectively, and their combination yielded synergistic antitumor efficacy indicated by 50.0% (6/12) of complete tumor remission. Analyses of TILs revealed that anti-PD-1 increased CD8+ T cells, while MSC_IL-12 led to infiltration of CD4+ T cells and NK cells. Both therapies reduced frequencies of Tregs. All these aspects observed in each monotherapy group were superimposed in the combination group. Notably, no tumor growth was observed upon rechallenge in cured mice, indicating long-term immunity against glioblastoma provoked by the therapies. Single-cell RNA-seq data confirmed these results and revealed that the combined treatment led to immune-favorable tumor microenvironment-CD4+, CD8+ T cells, effector memory T cells, and activated microglia were increased, whereas exhausted T cells, Tregs, and M2 polarized microglia were reduced. CONCLUSION: Anti-PD-1 and MSC_IL-12 monotherapies show long-term therapeutic responses, and their combination further enhances antitumor efficacy against glioblastoma via inducing immune-favorable tumor microenvironment.

Mutational Landscape of Normal Human Skin: Clues to Understanding Early-Stage Carcinogenesis in Keratinocyte Neoplasia.

Normal skin contains numerous clones carrying cancer driver mutations. However, the mutational landscape of normal skin and its clonal relationship with skin cancer requires further elucidation. The aim of our study was to investigate the mutational landscape of normal human skin. We performed whole-exome sequencing using physiologically normal skin tissues and the matched peripheral blood (n = 39) and adjacent-matched skin cancers from a subset of patients (n = 10). Exposed skin harbored a median of 530 mutations (10.4/mb, range = 51-2,947), whereas nonexposed skin majorly exhibited significantly fewer mutations (median = 13, 0.25/mb, range = 1-166). Patient age was significantly correlated with the mutational burden. Mutations in six driver genes (NOTCH1, FAT1, TP53, PPM1D, KMT2D, and ASXL1) were identified. De novo mutational signature analysis identified a single signature with components of UV- and aging-related signatures. Normal skin harbored only three instances of copy-neutral loss of heterozygosity in 9q (n = 2) and 6q (n = 1). The mutational burden of normal skin was not correlated with that of matched skin cancers, and no protein-coding mutations were shared. In conclusion, we revealed the mutational landscape of normal skin, highlighting the role of driver genes in the malignant progression of normal skin.

Single-cell and spatial sequencing application in pathology.

Traditionally, diagnostic pathology uses histology representing structural alterations in a disease's cells and tissues. In many cases, however, it is supplemented by other morphology-based methods such as immunohistochemistry and fluorescent in situ hybridization. Single-cell RNA sequencing (scRNA-seq) is one of the strategies that may help tackle the heterogeneous cells in a disease, but it does not usually provide histologic information. Spatial sequencing is designed to assign cell types, subtypes, or states according to the mRNA expression on a histological section by RNA sequencing. It can provide mRNA expressions not only of diseased cells, such as cancer cells but also of stromal cells, such as immune cells, fibroblasts, and vascular cells. In this review, we studied current methods of spatial transcriptome sequencing based on their technical backgrounds, tissue preparation, and analytic procedures. With the pathology examples, useful recommendations for pathologists who are just getting started to use spatial sequencing analysis in research are provided here. In addition, leveraging spatial sequencing by integration with scRNA-seq is reviewed. With the advantages of simultaneous histologic and single-cell information, spatial sequencing may give a molecular basis for pathological diagnosis, improve our understanding of diseases, and have potential clinical applications in prognostics and diagnostic pathology.

Genomic landscape of multiple Bowen's disease using whole-exome sequencing.

The genomic landscape of Bowen's disease (BD), with multiple manifestations, has not yet been determined. This study aimed to investigate the genomic alterations in multiple BD. We performed whole-exome sequencing of BD lesions (n = 9) and matched germlines collected from three patients with multiple (≥3) BD to detect somatic and germline mutations. We found a median of 64 somatic mutations in each sample (range 20-267). UV-signature mutations accounted for 64.9% (median, range 26.0%-82.1%) of point mutations. Putative driver mutations were found in five BDs (RB1 p.R445*, ARID2 p.R274*, TP53 p.Y163D/p.Y205D/p.R342*, KMT2C p.R4549C) but not in the other four lesions. Somatic mutations were not shared between multiple BD lesions collected from the same patient, indicating a different clonal origin. We also found no known pathogenic germline mutations in cancer-related genes. The mutational signature analysis revealed that UV signatures (SBS7a/7b) and age-related signatures (SBS1/5) were the main active signatures. Copy number alterations (CNAs) were found in two BDs: one with extensive CNA regions (21.7% of the genome), including driver genes (PIK3CA/SOX2/TP63 and MYC gain, and CDKN2A loss), and the other with 1q gain. Our study revealed that multiple BD lesions harbor distinct genomic landscapes, suggesting that they have different risks of malignant progression.

Whole-exome sequencing of secondary tumors arising from nevus sebaceous revealed additional genomic alterations besides RAS mutations.

Nevus sebaceous (NS) is a congenital hamartoma associated with an increased risk of secondary neoplasms in approximately 10%-20% of patients. However, additional genomic alterations underlying tumorigenesis in NS lesions have not been clarified. We performed whole-exome sequencing of archived tumor tissues (n = 8; six basal cell carcinomas and two trichoepitheliomas) and matched germline tissues (n = 7) with from seven patients with secondary tumors arising from NS. We also analyzed NS lesions without secondary tumors (n = 8). Somatic mutations and copy number alterations (CNAs) were analyzed. We identified a median of 129 somatic mutations (corresponding to 2.6/Mb in target regions, range 26-336) for eight tumors, while a median of 118 somatic mutations (2.3/Mb, range 1-196) for eight NS lesions. Known RAS hotspot mutations were found in seven of the eight tumors (six for HRAS p.G13R and one for HRAS p.Q61R) and in six of the eight NS lesions (four for HRAS p.G13R, one for KRAS p.G12C, and one KRAS p.G12D). Except RAS mutations, several putative driver mutations were detected in tumors: TP53 p.F134L/p.R213*, MYCN p.P59L, OR2Z1 p.P167S, PTPN14 p.Q768*, and SMO p.W535L. As for CNAs, two tumors harbored copy-loss in regions encompassing PTCH1 gene. However, eight NS lesions did not harbor both putative driver mutations and CNAs. In conclusion, our study revealed that secondary tumors arising from NS harbor known RAS hotspot mutations and additional genomic alterations, including putative driver mutations and PTCH1 copy-loss. These results could help to define the high-risk group for tumor development in patients with NS and provide evidence for prophylactic resection.

Striatal Hyperperfusion Observed in Dual-Phase 18F-FP-CIT PET Imaging of Hyperglycemic Chorea.

ABSTRACT: A 76-year-old woman with a history of diabetes mellitus presented with right-side dominant generalized chorea. At presentation, her blood glucose level was 500 mg/dL with an HbA1C of 11%. Because the patient had been on levodopa treatment from her primary physician, a dual-phase 18F-FP-CIT PET scan was performed. The early-phase images showed increased perfusion in the bilateral striatum, and the delayed-phase images revealed decreased uptake in the left caudate. Hyperperfusion in the striatum may indicate the acute phase of hyperglycemic chorea. This image illustrates the advantage of adding early-phase scans in 18F-FP-CIT PET in differentiating various hyperkinetic and hypokinetic disorders.

Genomic comparison between an in vitro three-dimensional culture model of melanoma and the original primary tumor.

Three-dimensional (3D) melanoma culture is a personalized in vitro model that can be used for high-fidelity pre-clinical testing and validation of novel therapies. However, whether the genomic landscape of 3D cultures faithfully reflects the original primary tumor which remains unknown. The purpose of our study was to compare the genomic landscapes of 3D culture models with those of the original tumors. Patient-derived xenograft (PDX) tumors were established by engrafting fresh melanoma tissue from each patient. Then, a 3D culture model was generated using cryopreserved PDX tumors embedded in pre-gelled porcine skin decellularized extracellular matrix with normal human dermal fibroblasts. Using whole-exome sequencing, the genomic landscapes of 3D cultures, PDX tumors, and the original tumor were compared. We found that 91.4% of single-nucleotide variants in the original tumor were detected in the 3D culture and PDX samples. Putative melanoma driver mutations (BRAF p.V600E, CDKN2A p.R7*, ADAMTS1 p.Q572*) were consistently identified in both the original tumor and 3D culture samples. Genome-wide copy number alteration profiles were almost identical between the original tumor and 3D culture samples, including the driver events of ARID1B loss, BRAF gain, and CCND1 gain. In conclusion, our study revealed that the genomic profiles of the original tumor and our 3D culture model showed high concordance, indicating the reliability of our 3D culture model in reflecting the original characteristics of the tumor.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells.

BACKGROUND: Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. OBJECTIVE: In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. METHODS: We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. RESULTS: Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. CONCLUSION: The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis.

Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.