Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-Nitroso- N -Pivaloyl- d -Penicillamine: Implications for Improved Diastolic Function and Cardiac Performance.

ABSTRACT: We previously reported a novel compound called S-nitroso- N -pivaloyl- d -penicillamine (SNPiP), which was screened from a group of nitric oxide donor compounds with a basic chemical structure of S-nitroso- N -acetylpenicillamine, to activate the nonneuronal acetylcholine system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The nonneuronal acetylcholine-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 hours after SNPiP administration) revealed that SNPiP initially induced Wnt and cyclic guanosine monophosphate-protein kinase G signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in response to SNPiP treatment, resulting in a metabolic shift from oxidative phosphorylation to glycolysis, which was suggested by reduced cardiac glucose contents while maintaining adenosine tri-phosphate levels. In addition, SNPiP significantly upregulated atrial natriuretic peptide and sarcolipin, which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

PTEN-induced kinase 1 gene single-nucleotide variants as biomarkers in adjuvant chemotherapy for colorectal cancer: a retrospective study.

BACKGROUND: Fluoropyrimidine-based postoperative adjuvant chemotherapy is globally recommended for high-risk stage II and stage III colon cancer. However, adjuvant chemotherapy is often associated with severe adverse events and is not highly effective in preventing recurrence. Therefore, discovery of novel molecular biomarkers of postoperative adjuvant chemotherapy to identify patients at increased risk of recurrent colorectal cancer is warranted. Autophagy (including mitophagy) is activated under chemotherapy-induced stress and contributes to chemotherapy resistance. Expression of autophagy-related genes and their single-nucleotide polymorphisms are reported to be effective predictors of chemotherapy response in some cancers. Our goal was to evaluate the relationship between single-nucleotide variants of autophagy-related genes and recurrence rates in order to identify novel biomarkers that predict the effect of adjuvant chemotherapy in colorectal cancer. METHODS: We analyzed surgical or biopsy specimens from 84 patients who underwent radical surgery followed by fluoropyrimidine-based adjuvant chemotherapy at Saitama Medical University International Medical Center between January and December 2016. Using targeted enrichment sequencing, we identified single-nucleotide variants and insertions/deletions in 50 genes, including autophagy-related genes, and examined their association with colorectal cancer recurrence rates. RESULTS: We detected 560 single-nucleotide variants and insertions/deletions in the target region. The results of Fisher's exact test indicated that the recurrence rate of colorectal cancer after adjuvant chemotherapy was significantly lower in patients with the single-nucleotide variants (c.1018G > A [p < 0.005] or c.1562A > C [p < 0.01]) of the mitophagy-related gene PTEN-induced kinase 1. CONCLUSIONS: The two single-nucleotide variants of PINK1 gene may be biomarkers of non-recurrence in colorectal cancer patients who received postoperative adjuvant chemotherapy.

Vimentin-positive invasive breast carcinoma of no special type: A breast carcinoma with lethal biological characteristics.

Vimentin is a stable mesenchymal immunohistochemical marker and is widely recognized as a major marker of mesenchymal tumors. The purpose of the present study was to investigate if the vimentin expression status might serve as a significant predictor of outcomes in patients with invasive breast carcinoma of no special type (IBC-NST) and to investigate, by comprehensive RNA sequencing analyses, the mechanisms involved in the heightened malignant potential of vimentin-positive IBC-NSTs. This study, conducted using the data of 855 patients with IBC-NST, clearly identified vimentin expression status as a very important independent biological parameter for accurately predicting the outcomes in patients with IBC-NST. RNA sequence analyses clearly demonstrated significant upregulation of coding RNAs known to be closely associated with cell proliferation or cellular senescence, and significant downregulation of coding RNAs known to be closely associated with transmembrane transport in vimentin-positive IBC-NSTs. We conclude that vimentin-positive IBC-NSTs show heightened malignant biological characteristics, possibly attributable to the upregulation of RNAs closely associated with proliferative activity and cellular senescence, and downregulation of RNAs closely associated with transmembrane transport in IBC-NSTs.

Effects of Pparγ1 deletion on late-stage murine embryogenesis and cells that undergo endocycle.

Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.

Two DNA binding domains of MGA act in combination to suppress ectopic activation of meiosis-related genes in mouse embryonic stem cells.

Although the physiological meaning of the high potential of mouse embryonic stem cells (ESCs) for meiotic entry is not understood, a rigid safeguarding system is required to prevent ectopic onset of meiosis. PRC1.6, a non-canonical PRC1, is known for its suppression of precocious and ectopic meiotic onset in germ cells and ESCs, respectively. MGA, a scaffolding component of PRC1.6, bears two distinct DNA-binding domains termed bHLHZ and T-box. However, it is unclear how this feature contributes to the functions of PRC1.6. Here, we demonstrated that both domains repress distinct sets of genes in murine ESCs, but substantial numbers of meiosis-related genes are included in both gene sets. In addition, our data demonstrated that bHLHZ is crucially involved in repressing the expression of Meiosin, which plays essential roles in meiotic entry with Stra8, revealing at least part of the molecular mechanisms that link negative and positive regulation of meiotic onset.

Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m6A modification.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D-TLS interaction is essential for pncRNA-D-stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS-pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

Identification of the Coiled-Coil Domain as an Essential Methyl-CpG-Binding Domain Protein 3 Element for Preserving Lineage Commitment Potential of Embryonic Stem Cells.

Embryonic stem cells (ESCs) exhibit two salient features beneficial for regenerative medicine: unlimited self-renewal and pluripotency. Methyl-CpG-binding domain protein 3 (Mbd3), a scaffolding component of the nucleosome remodeling deacetylase complex, is a specific regulator of pluripotency, as ESCs lacking Mbd3 are defective for lineage commitment potential but retain normal self-renewal properties. However, functional similarities and dissimilarities among the three Mbd3 isoforms (a, b, and c) have not been intensively explored. Herein, we demonstrated that Mbd3c, which lacks an entire portion of the MBD domain, exerted equivalent activity for counteracting the defective lineage commitment potential of Mbd3-knockout ESCs. Our analyses also revealed that the coiled-coil domain common to all three MBD3 isoforms, but not the MBD domain, plays a crucial role in this activity. Mechanistically, our data demonstrate that the activity of the coiled-coil domain is exerted, at least in part, through recruitment of polycomb repressive complex 2 to a subset of genes linked to development and organogenesis, thus establishing stable transcriptional repression. Stem Cells 2018;36:1355-1367.

Does MAX open up a new avenue for meiotic research?

Meiosis is a central event of sexual reproduction. Like somatic cells, germ cells conduct mitosis to increase their cell number, but unlike somatic cells, germ cells switch their cell division mode from mitosis to meiosis at a certain point in gametogenesis. However, the molecular basis of this switch remains elusive. In this review article, we give an overview of the onset of mammalian meiosis, including our recent finding that MYC Associated Factor X (MAX) prevents ectopic and precocious meiosis in embryonic stem cells (ESCs) and germ cells, respectively. We present a hypothetical model of a MAX-centered molecular network that regulates meiotic entry in mammals and propose that inducible Max knockout ESCs provide an excellent platform for exploring the molecular mechanisms of meiosis initiation, while excluding other aspects of gametogenesis.

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci.

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.

Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells.

Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N-terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2-associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2-dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2-associated protein network contributes to overcoming reprogramming barriers.

Forced expression of Nanog or Esrrb preserves the ESC status in the absence of nucleostemin expression.

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in a plethora of functions including ribosomal biogenesis and maintenance of telomere integrity. In addition to its expression in cancerous cells, the NS gene is expressed in stem cells including embryonic stem cells (ESCs). Previous knockdown and knockout studies have demonstrated that NS is important to preserve the self-renewality and high expression levels of pluripotency marker genes in ESCs. Here, we found that forced expression of Nanog or Esrrb, but not other pluripotency factors, resulted in the dispensability of NS expression in ESCs. However, the detrimental phenotypes of ESCs associated with ablation of NS expression were not mitigated by forced expression of Rad51 or a nucleolar localization-defective NS mutant that counteracts the damage associated with loss of NS expression in other NS-expressing cells such as neural stem/progenitor cells. Thus, our results indicate that NS participates in preservation of the viability and integrity of ESCs, which is distinct from that in other NS-expressing cells.

Functional compensation between Myc and PI3K signaling supports self-renewal of embryonic stem cells.

c-Myc and phosphatidylinositol 3-OH kinase (PI3K) both participate in diverse cellular processes, including cell cycle control and tumorigenic transformation. They also contribute to preserving embryonic stem cell (ESC) characteristics. However, in spite of the vast knowledge, the molecular relationship between c-Myc and PI3K in ESCs is not known. Herein, we demonstrate that c-Myc and PI3K function cooperatively but independently to support ESC self-renewal when murine ESCs are cultured under conventional culture condition. Interestingly, culture of ESCs in 2i-condition including a GSK3ß and MEK inhibitor renders both PI3K and Myc signaling dispensable for the maintenance of pluripotent properties. These results suggest that the requirement for an oncogenic proliferation-dependent mechanism sustained by Myc and PI3K is context dependent and that the 2i-condition liberates ESCs from the dependence of this mechanism.

Similarity and dissimilarity in alterations of the gene expression profile associated with inhalational anesthesia between sevoflurane and desflurane.

Although sevoflurane is one of the most commonly used inhalational anesthetic agents, the popularity of desflurane is increasing to a level similar to that of sevoflurane. Inhalational anesthesia generally activates and represses the expression of genes related to xenobiotic metabolism and immune response, respectively. However, there has been no comprehensive comparison of the effects of sevoflurane and desflurane on the expression of these genes. Thus, we used a next-generation sequencing method to compare alterations in the global gene expression profiles in the livers of rats subjected to inhalational anesthesia by sevoflurane or desflurane. Our bioinformatics analyses revealed that sevoflurane and, to a greater extent, desflurane significantly activated genes related to xenobiotic metabolism. Our analyses also revealed that both anesthetic agents, especially sevoflurane, downregulated many genes related to immune response.

Sirt1, p53, and p38(MAPK) are crucial regulators of detrimental phenotypes of embryonic stem cells with Max expression ablation.

c-Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. c-Myc is also an important regulator of self-renewal and pluripotency of embryonic stem cells (ESCs). We recently demonstrated that loss of the Max gene, encoding the best characterized partner for all Myc family proteins, causes loss of the pluripotent state and extensive cell death in ESCs strictly in this order. However, the mechanisms and molecules that are responsible for these phenotypes remain largely obscure. Here, we show that Sirt1, p53, and p38(MAPK) are crucially involved in the detrimental phenotype of Max-null ESCs. Moreover, our analyses revealed that these proteins are involved at varying levels to one another in the hierarchy of the pathway leading to cell death in Max-null ESCs.

Complete response to pembrolizumab in a patient with recurrent and metastatic urothelial bladder carcinoma reflecting coexisting sarcomatoid subtype and glandular differentiation: a case report.

In advanced urothelial carcinoma (UC), approximately 20% of patients respond to pembrolizumab, an anti-programmed cell death-1 (PD-1) antibody. Herein, we reported a single case of UC showing coexistence of sarcomatoid subtype and glandular differentiation. Notably, only glandular differentiation was recurrent, probably progressive, and metastatic, which showed complete response to pembrolizumab. An 80-year-old woman presented with hematuria and dysuria, and an intra-vesical tumor was detected on ultrasound. Transurethral resections (TUR) were performed three times. In the first TUR, a sub-pedunculated tumor and a flat lesion were closely but independently located. Pathologically, the sub-pedunculated tumor was an invasive UC, sarcomatoid subtype. Meanwhile, the flat lesion was invasive UC with glandular differentiation. Despite the second and the additional TUR, the tumor was growing and a lymph node metastasis was detected. The third TUR specimen showed UC with glandular differentiation, and a positive PD-L1 expression as well as high density CD8-positive lymphocytic cells infiltration were observed. Pembrolizumab was administered for four courses after terminating the chemotherapy. The CT scan revealed shrinkage of both primary tumor and metastases. Cystectomy and lymph nodes dissection were performed, and no residual carcinoma was detected. The therapeutic effect was regarded as pathological complete response. Pembrolizumab could be effective for special subtype or divergent differentiation of UC, particularly in an event of an 'immune hot' tumor. Supplementary Information: The online version contains supplementary material available at 10.1007/s13691-022-00568-5.

Temporal inhibition of the electron transport chain attenuates stress-induced cellular senescence by prolonged disturbance of proteostasis in human fibroblasts.

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.

Identification of novel oncogenes in oral cancer among elderly nonsmokers.

OBJECTIVES: In recent years, an increase in oral cancer among elderly nonsmokers has been noted. The aim of this study was to identify novel oncogenes in oral cancer in older nonsmokers. MATERIAL AND METHODS: Whole-exome sequencing (WES) data from 324 oral cancer patients were obtained from The Cancer Genome Atlas. Single nucleotide variants (SNVs) and insertions/deletions (INDELs) were extracted from the WES data of older patients. Fisher's exact test was performed to determine the specificity of variants in these genes. Finally, SNVs and INDELs were identified by target enrichment sequencing. RESULTS: Gene ontology analysis of 112 genes with significant SNVs or INDELs in nonsmokers revealed that nonsynonymous SNVs in HECTD4 were significantly more frequent in nonsmokers than in smokers by target enrichment sequencing (p = .02). CONCLUSIONS: Further investigation of the function of HECTD4 variants as oncogenes in older nonsmokers is warranted.