The germ cell-specific TAP-like protein NXF-2 forms a novel granular structure and is required for tra-2 3'UTR-dependent mRNA export in Caenorhabditis elegans.

TAP is a general mRNA export receptor and is highly conserved among eukaryotes. The nematode Caenorhabditis elegans has another TAP-like protein, NXF-2, but little is known about its function. In this study, we show that NXF-2 is specifically expressed in germ cells and forms a novel granular structure that is different from that of P granules and that NXF-2 granules are anchored to the nuclear periphery in the mitotic region of the hermaphrodite gonad. In contrast, NXF-2 granules are released within the whole cytoplasm in the meiotic region, where the feminization gene tra-2 starts to function. Both inhibition of XPO-1 (an ortholog of the export receptor CRM1) and mutation of the nuclear export signal of NXF-2 caused the release of NXF-2 granules from the nuclear periphery, indicating that anchoring of NXF-2 granules depends on XPO-1 function. Moreover, inhibition of NXF-2 resulted in a substantial nuclear accumulation of the reporter mRNA carrying the tra-2 3'UTR. These results suggest that, together with XPO-1, NXF-2 exports and anchors tra-2 mRNA to the nuclear periphery to avoid precocious translation until the germ cells reach the meiotic region, thereby contributing to the regulation of tra-2 mRNA expression.

Localization of substance P (SP)-immunoreactivity in the myenteric plexus of the rat esophagus.

The present immunohistochemical study was performed to examine the number, distribution, and chemical coding of intrinsic substance P (SP) neurons and nerve fibers within the esophagus and discuss their functional roles. Many SP neurons and nerve fibers were found in the myenteric plexus, and the SP neurons gradually decreased from the oral side toward the aboral side of the esophagus. Double-immunolabeling showed that most SP neurons were cholinergic (positive for choline acetyltransferase), and few were nitrergic (positive for nitric oxide synthase). Some cholinergic SP nerve terminals surrounded cell bodies of several myenteric neurons. In the muscularis mucosa and lower esophageal sphincter, and around blood vessels, numerous SP nerve endings were present, and many of them were cholinergic. Also, SP nerve endings were found on only a few motor endplates of the striated muscles, and most of them were calcitonin gene-related peptide (CGRP)-positive. Retrograde tracing using Fast Blue (FB) showed that numerous sensory neurons in the dorsal root ganglia (DRGs) and nodose ganglion (NG) projected to the esophagus, and most FB-labeled SP neurons were CGRP-positive. These results suggest that the intrinsic SP neurons in the rat esophagus may play roles as, at least, motor neurons, interneurons, and vasomotor neurons, which are involved in local regulation of smooth muscle motility, neuronal transmission, and blood circulation, respectively. Moreover, SP nerve endings on only a minority of motor endplates may be extrinsic, derived from DRGs or NG, and possibly detect chemical circumstances within motor endplates to modulate esophageal motility.

Computational Screening and Experimental Validation of Inhibitor Targeting the Complex Formation of Grb14 and Insulin Receptor.

The development of drugs targeting gene products associated with insulin resistance holds the potential to enhance our understanding of type 2 diabetes mellitus (T2DM). The virtual screening, based on a three-dimensional (3D) protein structure, is a potential technique to accelerate the development of molecular target drugs. Among the targets implicated in insulin resistance, the genetic characterization and protein function of Grb14 have been clarified without contradiction. The Grb14 gene displays significant variations in T2DM, and its gene product is known to inhibit the function of the insulin receptor (IR) by directly binding to the tyrosine kinase domain. In the present study, a virtual screening, based on a 3D structure of the IR tyrosine kinase domain (IRß) in complex with part of Grb14, was conducted to find compounds that can disrupt the complex formation between Grb14 and IRß. First, ten compounds were selected from 154,118 compounds via hierarchical in silico structure-based drug screening, composed of grid docking-based and genetic algorithm-based programs. The experimental validations suggested that the one compound can affect the blood glucose level. The molecular dynamics simulations and co-immunoprecipitation analysis showed that the compound did not completely suppress the protein-protein interaction between Grb14 and IR, though competitively bound to IR with the tyrosine kinase pseudosubstrate region in Grb14.

Complex Formation of Heme Oxygenase-2 with Heme Is Competitively Inhibited by the Cytosolic Domain of Caveolin-1.

The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82-101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1-101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

Risk and prognostic factors of post-catheterization pseudoaneurysm.

PURPOSE: To evaluate the risk and prognostic factors of post-catheterization pseudoaneurysm (PPA). MATERIAL AND METHODS: To identify the risk factors for PPA occurrence, clinical findings were compared between 22 consecutive patients with radiologically confirmed PPAs (PPA group) and 300 randomly extracted patients without PPA, who underwent transarterial angiography or intervention (sample group) between 1 January 2015 and 31 March 2020. The PPA group was further divided into those treated successfully with mechanical compression (group A) and those requiring ultrasound-guided thrombin injection after compression failed (group B). Univariate and multivariate analyses were used to compare patient demographics, preoperative laboratory findings, procedure details, PPA diameter, and time interval between the procedure and compression between groups A and B to evaluate the prognostic factors of PPA. RESULTS: The PPA group demonstrated significantly elevated prothrombin time international normalized ratios (PT/INR) (odds ratio [OR]: 6.27, 95% confidence interval [CI]: 2.020-19.5; p = 0.00151) and more frequent popliteal access (OR: 14.2, 95% CI: 1.040-195.0; p = 0.0467) compared to the sample group, and radial access decreased the risk of PPA (OR: 0.382, 95% CI: 0.0148-0.987; p = 0.0468). One of the 22 PPAs resolved spontaneously, and 11 others (52.4%) were successfully treated by mechanical compression. An interval exceeding 24 hours between the procedure and compression was the only significant prognostic factor (p = 0.0281) between groups A and B. CONCLUSIONS: Elevated PT/INR and popliteal access may predispose patients to PPA; close consideration of the site of access may lower the risk of refractory PPA.

MRG-1 is required for both chromatin-based transcriptional silencing and genomic integrity of primordial germ cells in Caenorhabditis elegans.

In Caenorhabditis elegans, germline cells remain transcriptionally silenced during embryogenesis. The transcriptional silencing is achieved by two different mechanisms: One is the inhibition of RNA polymerase II in P2-P4 cells at the establishment stage, and another is chromatin-based silencing in two primordial germ cells (PGCs) at the maintenance stage; however, the molecular mechanism underlying chromatin-based silencing is less understood. We investigated the role of the chromodomain protein MRG-1, which is an essential maternal factor for germline development, in transcriptional silencing in PGCs. PGCs lacking maternal MRG-1 showed increased levels of two histone modifications (H3K4me2 and H4K16ac), which are epigenetic markers for active transcription, and precocious activation of germline promoters. Loss of MES-4, a H3K36 methyltransferase, also caused similar derepression of the germline genes in PGCs, suggesting that both MRG-1 and MES-4 function in chromatin-based silencing in PGCs. In addition, the mrg-1 null mutant showed abnormal chromosome structures and a decrease in homologous recombinase RAD-51 foci in PGCs, but the mes-4 null mutant did not show such phenotypes. Taken together, we propose that MRG-1 has two distinct functions: chromatin-based transcriptional silencing and preserving genomic integrity at the maintenance stage of PGCs.

Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria.

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation.

The physical interaction of the human growth factor receptor-bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser358 , Ser362 , and Ser366 in hGrb14 by glycogen synthase kinase-3 repressed hGrb14-IR complex formation. In this study, we investigated phosphatase-mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14-derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser358 and Ser362 . Furthermore, coimmunoprecipitation experiments suggested that insulin-induced hGrb14-IR complex formation was repressed by the substitution of Ser358 or Ser362 with glutamic acid. These findings suggested that phosphate groups on Ser358 and Ser362 in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14-IR complex formation.

[Analysis of Workflow and Structuring of the Problem Element for Workflow Database Construction of Radiological Technologists in Radiation Therapy].

The work of radiological technologists is changing and more complicated because of the development of medical technology and implementation of information technology (IT). Although the cases of incident and accident have been reported, they have not been comprehensively analyzed in the workflow for radiotherapy. In this study, we visualized the workflow of radiological technologists in radiotherapy and revealed the causes of incidents and accidents. The work process was visualized by drawing workflow map. The structuring of problem was performed with interpretive structural modeling (ISM) method based on graph theory by analyzing of work categorized by safety management. Our results may be able to clarify the work of radiological technologists leads to the reduction of incidents and accidents in radiation therapy.

[Survey of the Requested Function for Quality Assurance System for Images (Kenzo System)].

As the use of filmless examination images, using various systems, has increased, and became common to perform KAKUTEI and save the images. In particular, the use of quality assurance system for images (Kenzo system) has increased to ensure the efficient performance of confirmed image. However, there has been no report showing what kind of function should be used or how to write the specifications of such a function in introducing the Kenzo system. Therefore, this study conducted a survey to the in-charge medical staff of medical institutions to provide "information included in the specifications when introducing medical systems". As a result, it is possible, through analyzing and clarifying the necessary functions of the Kenzo system, to apply it in medical institutions with various scales and workflows. The results indicate the person in charge was looking for functions, such as "coordination of information and image processing, securing the consistency of the information, and clarifying responsibility using the records of confirmed persons". We showed examples of how to describe these in the specifications.

DND protein functions as a translation repressor during zebrafish embryogenesis.

Germline and somatic cell distinction is regulated through a combination of microRNA and germ cell-specific RNA-binding proteins in zebrafish. An RNA-binding protein, DND, has been reported to relieve the miR-430-mediated repression of some germ plasm mRNAs such as nanos3 and tdrd7 in primordial germ cells (PGCs). Here, we showed that miR-430-mediated repression is not counteracted by the overexpression of DND protein in somatic cells. Using a λN-box B tethering assay in the embryo, we found that tethering of DND to reporter mRNA results in translation repression without affecting mRNA stability. Translation repression by DND was not dependent on another germline-specific translation repressor, Nanos3, in zebrafish embryos. Moreover, our data suggested that DND represses translation of nanog and dnd mRNAs, whereas an RNA-binding protein DAZ-like (DAZL) promotes dnd mRNA translation. Thus, our study showed that DND protein functions as a translation repressor of specific mRNAs to control PGC development in zebrafish.

Control of the heat stress-induced alternative splicing of a subset of genes by hnRNP K.

Pre-mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre-mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA-binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K-dependent manner, whereas hnRNP K is not necessary for the stress-induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA-binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.

The ELAV protein HuD stimulates cap-dependent translation in a Poly(A)- and eIF4A-dependent manner.

The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3' poly(A) tail and a 5' m(7)G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.

Carcinoma Ex Pleomorphic Adenoma of the Tongue: Difficulty in Diagnosis Between Metastasis of Breast Cancer and Salivary Tumor.

Carcinoma ex pleomorphic adenoma (CEPA) is a carcinoma that shows histologic evidence of arising in or from a benign pleomorphic adenoma. Carcinoma ex pleomorphic adenoma often occurs in parotid glands, but is extremely rarely in the tongue. A 53-year-old Japanese woman was referred to the Department of Oral and Maxillofacial Surgery, Nagasaki University Hospital, because of tumor of the right dorsum tongue. She had a history of surgery of breast cancer (invasive ductal carcinoma) and it was disseminated to the lung and bone. Macroscopic examination revealed an oval tumor with a smooth mucosal surface of 10 mm in diameter in the right dorsum tongue. A clinical diagnosis was metastasis from breast cancer or primary salivary gland tumor. Histologic diagnosis of the biopsy specimen was CEPA. She underwent partial glossectomy under general anesthesia. The final diagnosis of surgical materials was CEPA based on the differential diagnosis from breast carcinoma. She is alive bearing disseminated breast carcinoma without recurrence of CEPA at 6 months after glossectomy.

Propensity-Score Matched Comparison of Midterm Angiographic Outcomes of Sirolimus- Versus Everolimus- and Biolimus-Eluting Stents for De Novo Coronary Stenosis.

We conducted propensity-score matched comparisons of midterm angiographic outcomes of sirolimus (SES) versus either everolimus- (EES) or biolimus- (BES) eluting stents after placements for coronary stenosis in a daily practice environment since previous randomized trials did not demonstrate the superiority of EES and BES over SES in terms of midterm angiographic outcomes.The present study was a non-randomized, retrospective, and lesion-based study, recruiting angiographically followed-up lesions within 550 days after successful and elective SES (n = 1793), EES (n = 1303), or BES (n = 324) placement for de novo native coronary stenosis during the period from August 2004 to January 2014 at 6 institutes. The endpoint, as an angiographic surrogate marker of clinical efficacy, was the distribution of in-stent follow-up percent diameter stenosis (%DS) which comprised the percentages of 1) follow-up %DS < 20 and 2) follow-up %DS > 50. Propensityscore matched analyses were conducted to adjust 21 baselines.In 1215 baseline adjusted lesions, the endpoints in the EES group [1) 74.1%, and 2) 4.6%] were significantly different from those in the SES group [57.9%; P < 0.001, 7.2%; P = 0.006, respectively). In 307 baseline adjusted lesions, the endpoints in the BES group [1) 80.5%, 2) 2.0%] were significantly different from those in the SES group [59.3%; P < 0.001, 2) 8.1%; P = 0.001, respectively].The present study is the first to confirm the superiority of midterm angiographic outcomes after the placement of EES and BES over SES for de novo coronary stenosis in a clinical setting.

Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality.

Interaction and colocalization of HERMES/RBPMS with NonO, PSF, and G3BP1 in neuronal cytoplasmic RNP granules in mouse retinal line cells.

HERMES, also called RBPMS, is a conserved RNA binding protein with a single RNA recognition motif (RRM) that is abundantly expressed in retinal ganglion cells (RGCs) and in the heart in vertebrates. Here, we identified NonO and PSF as the interacting proteins of HERMES only when the neuronal differentiation of the retinal cell line RGC-5 was induced. Although NonO and PSF are nuclear paraspeckle components, these proteins formed cytoplasmic granules with HERMES in the neurites. G3BP1, a component of stress granules, was also colocalized to the granules, interacting with NonO and HERMES even in the absence of cellular stress. Consistent with a previous report that KIF5 interacts with neuronal granules, the localization of KIF5A overlapped with the cytoplasmic granules in differentiated RGC-5 cells. Thus, our study strongly suggests that the cytoplasmic granule containing HERMES, NonO, PSF, and G3BP1 is a neuronal RNA-protein granule that is transported in neurites during retinal differentiation.

Determining c-Myb protein levels can isolate functional hematopoietic stem cell subtypes.

The transcription factor c-Myb was originally identified as a transforming oncoprotein encoded by two avian leukemia viruses. Subsequently, through the generation of mouse models that affect its expression, c-Myb has been shown to be a key regulator of hematopoiesis, including having critical roles in hematopoietic stem cells (HSCs). The precise function of c-Myb in HSCs although remains unclear. We have generated a novel c-myb allele in mice that allows direct observation of c-Myb protein levels in single cells. Using this reporter line we demonstrate that subtypes of HSCs can be isolated based upon their respective c-Myb protein expression levels. HSCs expressing low levels of c-Myb protein (c-Myb(low) HSC) appear to represent the most immature, dormant HSCs and they are a predominant component of HSCs that retain bromodeoxyuridine labeling. Hematopoietic stress, induced by 5-fluorouracil ablation, revealed that in this circumstance c-Myb-expressing cells become critical for multilineage repopulation. The discrimination of HSC subpopulations based on c-Myb protein levels is not reflected in the levels of c-myb mRNA, there being no more than a 1.3-fold difference comparing c-Myb(low) and c-Myb(high) HSCs. This illustrates how essential it is to include protein studies when aiming to understand the regulatory networks that control stem cell behavior.

Discovery of a novel and conserved Plasmodium falciparum exported protein that is important for adhesion of PfEMP1 at the surface of infected erythrocytes.

Plasmodium falciparum virulence is linked to its ability to sequester in post-capillary venules in the human host. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the main variant surface antigen implicated in this process. Complete loss of parasite adhesion is linked to a large subtelomeric deletion on chromosome 9 in a number of laboratory strains such as D10 and T9-96. Similar to the cytoadherent reference line FCR3, D10 strain expresses PfEMP1 on the surface of parasitized erythrocytes, however without any detectable cytoadhesion. To investigate which of the deleted subtelomeric genes may be implicated in parasite adhesion, we selected 12 genes for D10 complementation studies that are predicted to code for proteins exported to the red blood cell. We identified a novel single copy gene (PF3D7_0936500) restricted to P. falciparum that restores adhesion to CD36, termed here virulence-associated protein 1 (Pfvap1). Protein knockdown and gene knockout experiments confirmed a role of PfVAP1 in the adhesion process in FCR3 parasites. PfVAP1 is co-exported with PfEMP1 into the host cell via vesicle-like structures called Maurer's clefts. This study identifies a novel highly conserved parasite molecule that contributes to parasite virulence possibly by assisting PfEMP1 to establish functional adhesion at the host cell surface.

The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes.