Inhibition of protein arginine methyltransferase 6 activates interferon signaling and induces the apoptosis of endometrial cancer cells via histone modification.

Histone modification, a major epigenetic mechanism regulating gene expression through chromatin remodeling, introduces dynamic changes in chromatin architecture. Protein arginine methyltransferase 6 (PRMT6) is overexpressed in various types of cancer, including prostate, lung and endometrial cancer (EC). Epigenome regulates the expression of endogenous retrovirus (ERV), which activates interferon signaling related to cancer. The antitumor effects of PRMT6 inhibition and the role of PRMT6 in EC were investigated, using epigenome multi­omics analysis, including an assay for chromatin immunoprecipitation sequencing (ChIP­seq) and RNA sequencing (RNA­seq). The expression of PRMT6 in EC was analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and immunohistochemistry (IHC). The prognostic impact of PRMT6 expression was evaluated using IHC. The effects of PRMT6­knockdown (KD) were investigated using cell viability and apoptosis assays, as well as its effects on the epigenome, using ChIP­seq of H3K27ac antibodies and RNA­seq. Finally, the downstream targets identified by multi­omics analysis were evaluated. PRMT6 was overexpressed in EC and associated with a poor prognosis. PRMT6­KD induced histone hypomethylation, while suppressing cell growth and apoptosis. ChIP­seq revealed that PRMT6 regulated genomic regions related to interferons and apoptosis through histone modifications. The RNA­seq data demonstrated altered interferon­related pathways and increased expression of tumor suppressor genes, including NK6 homeobox 1 and phosphoinositide­3­kinase regulatory subunit 1, following PRMT6­KD. RT­qPCR revealed that eight ERV genes which activated interferon signaling were upregulated by PRMT6­KD. The data of the present study suggested that PRMT6 inhibition induced apoptosis through interferon signaling activated by ERV. PRMT6 regulated tumor suppressor genes and may be a novel therapeutic target, to the best of our knowledge, in EC.

Evolution of a surgical system using deep learning in minimally invasive surgery (Review).

Recently, artificial intelligence (AI) has been applied in various fields due to the development of new learning methods, such as deep learning, and the marked progress in computational processing speed. AI is also being applied in the medical field for medical image recognition and omics analysis of genomes and other data. Recently, AI applications for videos of minimally invasive surgeries have also advanced, and studies on such applications are increasing. In the present review, studies that focused on the following topics were selected: i) Organ and anatomy identification, ii) instrument identification, iii) procedure and surgical phase recognition, iv) surgery-time prediction, v) identification of an appropriate incision line, and vi) surgical education. The development of autonomous surgical robots is also progressing, with the Smart Tissue Autonomous Robot (STAR) and RAVEN systems being the most reported developments. STAR, in particular, is currently being used in laparoscopic imaging to recognize the surgical site from laparoscopic images and is in the process of establishing an automated suturing system, albeit in animal experiments. The present review examined the possibility of fully autonomous surgical robots in the future.

Development of a deep learning method for improving diagnostic accuracy for uterine sarcoma cases.

Uterine sarcomas have very poor prognoses and are sometimes difficult to distinguish from uterine leiomyomas on preoperative examinations. Herein, we investigated whether deep neural network (DNN) models can improve the accuracy of preoperative MRI-based diagnosis in patients with uterine sarcomas. Fifteen sequences of MRI for patients (uterine sarcoma group: n = 63; uterine leiomyoma: n = 200) were used to train the models. Six radiologists (three specialists, three practitioners) interpreted the same images for validation. The most important individual sequences for diagnosis were axial T2-weighted imaging (T2WI), sagittal T2WI, and diffusion-weighted imaging. These sequences also represented the most accurate combination (accuracy: 91.3%), achieving diagnostic ability comparable to that of specialists (accuracy: 88.3%) and superior to that of practitioners (accuracy: 80.1%). Moreover, radiologists' diagnostic accuracy improved when provided with DNN results (specialists: 89.6%; practitioners: 92.3%). Our DNN models are valuable to improve diagnostic accuracy, especially in filling the gap of clinical skills between interpreters. This method can be a universal model for the use of deep learning in the diagnostic imaging of rare tumors.

The Histone Methyltransferase SETD8 Regulates the Expression of Tumor Suppressor Genes via H4K20 Methylation and the p53 Signaling Pathway in Endometrial Cancer Cells.

The histone methyltransferase SET domain-containing protein 8 (SETD8), which methylates histone H4 lysine 20 (H4K20) and non-histone proteins such as p53, plays key roles in human carcinogenesis. Our aim was to determine the involvement of SETD8 in endometrial cancer and its therapeutic potential and identify the downstream genes regulated by SETD8 via H4K20 methylation and the p53 signaling pathway. We examined the expression profile of SETD8 and evaluated whether SETD8 plays a critical role in the proliferation of endometrial cancer cells using small interfering RNAs (siRNAs). We identified the prognostically important genes regulated by SETD8 via H4K20 methylation and p53 signaling using chromatin immunoprecipitation sequencing, RNA sequencing, and machine learning. We confirmed that SETD8 expression was elevated in endometrial cancer tissues. Our in vitro results suggest that the suppression of SETD8 using siRNA or a selective inhibitor attenuated cell proliferation and promoted the apoptosis of endometrial cancer cells. In these cells, SETD8 regulates genes via H4K20 methylation and the p53 signaling pathway. We also identified the prognostically important genes related to apoptosis, such as those encoding KIAA1324 and TP73, in endometrial cancer. SETD8 is an important gene for carcinogenesis and progression of endometrial cancer via H4K20 methylation.

DOSE AND DOSE-RATE DEPENDENCE OF DSB-TYPE MUTANTS INDUCED BY X-RAYS OR TRITIUM BETA-RAYS: AN APPROACH USING A HYPERSENSITIVE SYSTEM.

To evaluate biological effects triggered by low levels of radiation, we established a uniquely sensitive experimental system to detect somatic mutations. By using the system, we found that mutant frequencies induced by X-rays were statistically significant at doses over 0.15 Gy, and a linear dose relationship with the mutant frequency was observed at doses over 0.15 Gy. The mutation spectra analysis revealed that mutation events generated by X-ray doses below 0.1 Gy were similar to those observed in unirradiated controls. In addition, a significant inflection point for both, the mutant frequency and the mutation spectra, was found at dose-rates around 11 mGy/day when cells were cultured in medium containing tritiated water. Because induced radiation-type events presented a clear dose/dose-rate dependency above the critical dose or the inflection point, these observations suggest that mutation events generated by radiation could change at a threshold dose-rate or a critical dose.

Genetic diagnosis of pseudomyxoma peritonei originating from mucinous borderline tumor inside an ovarian teratoma.

BACKGROUND: Pseudomyxoma peritonei is a rare disease condition mainly caused by primary mucinous tumors from the appendix and rarely from the ovary, such as when mucinous ovarian tumors arise from within a teratoma. Molecular analyses of pseudomyxoma from the appendix showed that KRAS and GNAS pathogenic variants are common genetic features of pseudomyxoma peritonei. However, the origin of the tumors is difficult to be identified via genetic variants alone. This study presents a case of pseudomyxoma peritonei of ovarian origin, which was diagnosed by comprehensive genomic profiling with ploidy analysis in a series of primary, recurrent, and autopsy tumor specimens. CASE PRESENTATION: A 40-year-old woman was diagnosed with Stage IC2 mucinous ovarian tumor of borderline malignancy with mature cystic teratoma, upon clinical pathology. Immunohistochemical analysis suggested that the mucinous tumor was derived from the intestinal component of an ovarian teratoma. Three years later, intraperitoneal recurrence was detected, which subsequently progressed to pseudomyxoma peritonei. Genomic analysis detected KRAS (G12D), GNAS (R201C), and FBXW7 (R367*) variants in the primary tumor. In addition, the tumor showed aneuploidy with loss of heterozygosity (LOH) in all its chromosomes, which suggested that the primary ovarian tumor was derived from germ cells. Existence of one Barr body suggested the existence of uniparental disomy of the tumors throughout the genome, instead of a haploid genotype. All three pathogenic variants remained positive in the initial recurrent tumor, as well as in the paired DNA from the whole blood in pseudomyxoma peritonei. The pathogenic variant of KRAS (G12D) was also identified in the autopsy specimen of the appendix by droplet digital polymerase chain reaction. CONCLUSIONS: This study pathologically and genetically confirmed that the primary ovarian borderline tumor was derived from the intestinal component of an ovarian teratoma, and that the subsequent pseudomyxoma peritonei progressed from the primary ovarian tumor. Integrative genomic analysis was useful to identify cellular origin of tumors, as well as to precisely interpret the process of disease progression.

Histone arginine methyltransferase CARM1 selective inhibitor TP-064 induces apoptosis in endometrial cancer.

Histone modification is the key epigenetic mechanism that regulates gene expression. Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that catalyzes dimethylation of histone H3 (H3R17) at arginine 17. Lately, it has been suggested that CARM1 is associated with human carcinogenesis, and the CARM1-selective inhibitor, TP-064, has been shown to be a potential therapeutic agent for multiple myeloma. However, the physiological significance of CARM1 in endometrial cancer remains unclear. Therefore, we aimed to explore the role of CARM1 and the effect of TP-064 in endometrial cancer. To this end, we analyzed CARM1 expression in endometrial cancer using quantitative real-time polymerase chain reaction and examined the antitumor mechanism with CARM1 knockdown endometrial cancer cells. Moreover, we evaluated the therapeutic capability of TP-064 in endometrial cancer cells. CARM1 was remarkably overexpressed in 52 endometrial cancer tissues compared to normal endometrial tissues. The growth of CARM1 knockdown endometrial cancer cells was suppressed and CARM1 knockdown induced apoptosis. TP-064 also inhibited endometrial cancer cell growth and declined the number of endometrial cancer cell colonies. These data suggest that CARM1 may be a powerful therapeutic target for endometrial cancer.

Early postnatal nicotine exposure disrupts the α2* nicotinic acetylcholine receptor-mediated control of oriens-lacunosum moleculare cells during adolescence in rats.

Maternal cigarette smoking during pregnancy and maternal nicotine exposure in animal models are associated with cognitive impairments in offspring. However, the underlying mechanism remains unknown. Oriens-lacunosum moleculare (OLM) cells expressing α2* nicotinic acetylcholine receptors (nAChRs) are an important component of hippocampal circuitry, gating information flow and long-term potentiation (LTP) in the CA1 region. Here we investigated whether early postnatal nicotine exposure alters the normal role of α2*-nAChR-expressing OLM cells during adolescence in rats. We found that early postnatal nicotine exposure significantly decreased not only the number of α2-mRNA-expressing interneurons in the stratum oriens/alveus, but also α2*-nAChR-mediated responses in OLM cells. These effects of nicotine were prevented by co-administration with the nonselective nAChR antagonist mecamylamine, suggesting that nicotine-induced activation, but not desensitization, of nAChRs mediates the effects. α2*-nAChR-mediated depolarization of OLM cells normally triggers action potentials, causing an increase in spontaneous inhibitory postsynaptic currents in synaptically connected pyramidal cells. However, these α2*-nAChR-mediated effects were profoundly reduced after early postnatal nicotine exposure, suggesting altered control of CA1 circuits by α2*-nAChR-expressing OLM cells. Furthermore, these effects were associated with altered excitatory neural activity and LTP as well as the loss of normal α2*-nAChR-mediated control of excitatory neural activity and LTP. These findings suggest the altered function of α2*-nAChR-expressing OLM cells as an important target of further study for identifying the mechanisms underlying the cognitive impairment induced by maternal smoking during pregnancy.

Involvement of glutamate 97 in ion influx through photo-activated channelrhodopsin-2.

The light absorption of a channelrhodopsin-2 (ChR2) is followed by conformational changes to the molecule, which allows the channel structure to become permeable to cations. Previously, a single point mutation in ChR2, which replaces glutamate residue 97 with a nonpolar alanine (E97A), was found to attenuate the photocurrent, suggesting that the E97 residue is involved in ion flux regulation. Here, the significance of E97 and its counterpart ChR1 (E136) were extensively studied by mutagenesis, whereby we replaced these glutamates with aspartate (D), glutamine (Q) or arginine (R). We found that the charge at this position strongly influences ion permeation and that the photocurrents were attenuated in the order of ChR2>E97D≈E97Q>E97R. We observed similar results with our chimeric/synthetic/artificial construct, ChR-wide receiver (ChRWR), which contains the first to fifth transmembrane helices of ChR1. The E-to-Q or E-to-R mutations, but not the E-to-D mutation, strongly retarded the sensitivity to the Gd(3+)-dependent blocking of the ChR1 or ChR2 channels. Our results suggest that the glutamate residue at this position lies in the outer pore, where it interacts with a cation to facilitate dehydration, and that this residue is the primary binding target of Gd(3+).

Opto-current-clamp actuation of cortical neurons using a strategically designed channelrhodopsin.

BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state.