Excess microtubule and F-actin formation mediates shortening and loss of primary cilia in response to a hyperosmotic milieu.

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shock-induced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin.

Nairovirus polymerase mutations associated with the establishment of persistent infection in human cells.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne virus of the Orthonairovirus genus, persistently infects tick cells. It has been reported to establish persistent infection in non-human primates, but virological analysis has not yet been performed in human cells. Here, we investigated whether and how nairoviruses persistently infect human cells using Hazara orthonairovirus (HAZV), a surrogate model for CCHFV. We established a human cell line that was persistently infected with HAZV. Surprisingly, virions of persistently infected HAZV (HAZVpi) were not observed in the culture supernatants. There were five mutations (mut1, mut2, mut3, mut4, and mut5) in L protein of HAZVpi. Mutations in L protein of HAZVpi contribute to non-detection of virion in the supernatants. Lmut4 was found to cause low viral growth rate, despite its high polymerase activity. The low growth rate was restored by Lmut2, Lmut3, and Lmut5. The polymerase activity of Lmut1 was extremely low, and recombinant HAZV carrying Lmut1 (rHAZV/Lmut1) was not released into the supernatants. However, genomes of rHAZV/Lmut1 were retained in the infected cells. All mutations (Lmut1-5) found in L protein of HAZVpi were required for experimental reproduction of HAZVpi, and only Lmut1 and Lmut4 were insufficient. We demonstrated that point mutations in viral polymerase contribute to the establishment of persistent HAZV infection. Furthermore, innate immunity was found to be suppressed in HAZVpi-infected cells, which also potentially contributes to viral persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells. IMPORTANCE: We investigated whether and how nairoviruses persistently infect human cells, using Hazara orthonairovirus (HAZV), a surrogate model for Crimean-Congo hemorrhagic fever virus. We established a human cell line that was persistently infected with HAZV. Five mutations were found in L protein of persistently infected HAZV (HAZVpi): mut1, mut2, mut3, mut4, and mut5. Among them, Lmut1 and Lmut4 restricted viral growth by low polymerase activity and low growth rate, respectively, leading to inhibition of viral overgrowth. The restriction of viral growth caused by Lmut1 and Lmut4 was compensated by other mutations, including Lmut2, Lmut3, and Lmut5. Each of the mutations found in L protein of HAZVpi was concluded to cooperatively modulate viral growth, which facilitates the establishment of persistent infection. Suppression of innate immunity also potentially contributes to virus persistence. This is the first presentation of a possible mechanism behind how nairoviruses establish persistent infection in human cells.

Tumor-derived mitochondrial formyl peptides suppress tumor immunity through modification of the tumor microenvironment.

Mitochondrial N-formylpeptides are released from damaged or dead cells to the extracellular spaces and cause inflammatory responses. The role of mitochondrial N-formylpeptides in aseptic systemic inflammatory response syndromes induced by trauma or cardiac surgery has been well investigated. However, there are no reports regarding the role of mitochondrial N-formylpeptides in cancer. In this study, we investigated the role of tumor cell-derived mitochondrial N-formylpeptides in anti-tumor immunity using knockout murine tumor cells of mitochondrial methionyl-tRNA formyltransferase (MTFMT), which catalyze N-formylation of mitochondrial DNA-encoded proteins. There was no apparent difference among the wild-type and MTFMT-knockout clones of E.G7-OVA cells with respect to morphology, mitochondrial dynamics, glycolysis and oxidative phosphorylation, oxygen consumption rate, or in vitro cell growth. In contrast, in vivo tumor growth of MTFMT-knockout cells was slower than that of wild-type cells. A reduced number of myeloid-derived suppressor cells and an increase of cytotoxic T-lymphocytes in the tumor tissues were observed in the MTFMT-knockout tumors. These results suggested that tumor cell-derived mitochondrial N-formylpeptides had a negative role in the host anti-tumor immunity through modification of the tumor microenvironment.

Three-dimensional ultrastructural and anatomical analysis of prostatic neuroendocrine cells in mice.

BACKGROUND: A few studies have examined the ultrastructure of prostatic neuroendocrine cells (NECs), and no study has focused on their ultrastructure in three dimensions. In this study, three-dimensional ultrastructural analysis of mouse prostatic NECs was performed to clarify their anatomical characteristics. METHODS: Three 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with physiological saline and 2% paraformaldehyde, and then placed in 2.5% glutaraldehyde in 0.1 M cacodylate (pH 7.3) buffer for electron microscopy. After perfusion, the lower urinary tract, which included the bladder, prostate, coagulation gland, seminal vesicle, upper vas deferens, and urethra, was removed, and the specimen was cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on NECs, the surrounding cells, tissues, and nerves using focused ion beam/scanning electron microscope tomography. RESULTS: Twenty-seven serial sections were used in the present study, and 32 mouse prostatic NECs were analyzed. Morphologically, the NECs could be classified into three types: flask, flat, and closed. Closed-shaped NECs were always adjacent to flask-shaped cells. The flask-shaped and flat NECs were in direct contact with the ductal lumen and always had microvilli at their contact points. Many of the NECs had accompanying nerves, some of which terminated on the surface in contact with the NEC. CONCLUSIONS: Three-dimensional ultrastructural analysis of mouse prostatic NECs was performed. These cells can be classified into three types based on shape. Novel findings include the presence of microvilli at their points of contact with the ductal lumen and the presence of accompanying nerves.

The bipartite promoter of Orthonairovirus hazaraense large segment.

IMPORTANCE: The realization that segmented negative-strand RNA virus genome ribonucleoproteins are never free as their RNA ends are always bound to the viral polymerase has highlighted the problem of how these genome segments are replicated and express their mRNAs while their RNA ends remain associated with the polymerase throughout the cycles of RNA synthesis. This study of the length and nucleotide composition of the Orthonairovirus hazaraense L segment-specific double-stranded RNA (dsRNA) promoter element (the promoter duplex) provides insight into how its mRNA might be initiated and suggests that this promoter element acts via its separated single strands as well as via dsRNA.

Behavioral and neurological effects of Vrk1 deficiency in zebrafish.

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase, for which mutations have been reported cause to neurodegenerative diseases, including spinal muscular atrophy, characterized by microcephaly, motor dysfunction, and impaired cognitive function, in humans. Partial Vrk1 knockdown in mice has been associated with microcephaly and impaired motor function. However, the pathophysiological relationship between VRK1 and neurodegenerative disorders and the precise mechanism of VRK1-related microcephaly and motor function deficits have not been fully investigated. To address this, in this study, we established vrk1-deficient (vrk1-/-) zebrafish and found that they show mild microcephaly and impaired motor function with a low brain dopamine content. Furthermore, vrk1-/- zebrafish exhibited decreased cell proliferation, defects in nuclear envelope formation, and heterochromatin formation in the brain. To our knowledge, this is the first report demonstrating the important role of VRK1 in microcephaly and motor dysfunction in vivo using vrk1-/- zebrafish. These findings contribute to elucidating the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with microcephaly.

A Point Mutation in the Human Parainfluenza Virus Type 2 Nucleoprotein Leads to Two Separate Effects on Virus Replication.

Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), have lengths of precisely multiples-of-six nucleotides ("rule of six"), where each nucleoprotein subunit (NP) binds exactly six nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. The mutation of NPQ202 leads to two phenotypes: the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NPwt is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence of an rPIV2 NPQ202A prevented further study of the latter phenotype. By extensive and repeated cocultivation of transfected cells, an rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. IMPORTANCE Paramyxovirus genomes are contained within a noncovalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3' ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3' ends may play another role in the virus life cycle via their specific interaction with virus-modified cell membranes needed for the incorporation of viral NCs into budding virions.

Three-Dimensional Ultrastructural Analysis of the Head-Most Mitochondrial Roots of Mice Spermatozoa Using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) Tomography.

This study aimed to clarify the three-dimensional ultrastructure of head-side mice spermatozoa mitochondria. Six 13-week-old male C57BL/6 mice were deeply anesthetized, perfused with 2% paraformaldehyde, and placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for electron microscopy. After perfusion, the vas deferens was removed, and the specimens were cut into small cubes and subjected to postfixation and en bloc staining. Three-dimensional ultrastructural analysis was performed on five mitochondria on the spermatozoa head using conventional transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. Conventional TEM analysis showed that head-side mitochondria were not spiral in morphology but clearly horizontal to the sperm axis. However, this was difficult to evaluate further using conventional TEM. In the FIB/SEM analysis, the first and second head-most mitochondria were flat and straight, with no helix, and shaped as an attachment plug with two electrodes, and their tail side contacted the third mitochondrion. The third mitochondrion was shorter than the fourth and fifth and had a semicircular arching structure. The fourth and fifth mitochondria were spiral-shaped and intertwined. The redundant nuclear envelope encircled the head-most mitochondria. This ultrastructural analysis clarified that the head-most mitochondria have a unique morphology.

Three-Dimensional Ultrastructural and Volume Analysis of the Redundant Nuclear Envelope of Developing and Matured Sperm in Mice.

The ultrastructure of the nuclear envelope (NE) and redundant NE (RNE) of the spermatozoon cannot be observed in detail using conventional electron microscopy. Thus, this study aimed to employ transmission electron microscopy (TEM) and focused ion beam/scanning electron microscopy (FIB/SEM) tomography to fill this research gap. Male mice aged 13 weeks were deeply anesthetized, and the testes and vas deferens were extracted and processed for electron microscopy. In round spermatids, the acrosomal vesicle compressed the nucleus, and the acrosomal center was depressed. The nucleoli concentrated on the contralateral side of the acrosome formation site. In mature spermatozoa, the RNE accumulated in the neck with the residual bodies. The NE pores exhibited a hexagonal pattern. The body surface area and volume of the nuclei of spermatids and spermatozoa in each maturation phase were analyzed using FIB/SEM tomography. The body surface area and volume of the nuclei decreased during spermatid maturation into spermatozoa. The RNE converged at the sperm neck and possessed a honeycomb structure. The method used revealed that the nuclei of spermatids gradually condense as they mature into spermatozoa. This method may be used to analyze small tissues, such as RNE, and detect morphological abnormalities in microtissues, such as spermatozoa.

Immunoglobulin G deposition on proximal tubules and the tubular basement membrane in acute tubular injury complicated with focal segmental glomerulosclerosis (FSGS): A possible prediction tool for subclinical FSGS.

Immunofluorescent deposition of immunoglobulin G (IgG) in the tubular basement membrane (TBM) has been evaluated in the diagnosis of various diseases; however, few studies have investigated the immunofluorescence of acute tubular injury (ATI). Herein, we attempted to clarify IgG expression in the proximal tubular epithelium and TBM in ATI due to various causes. Patients with ATI with nephrotic-range proteinuria, including focal segmental glomerulosclerosis (FSGS, n = 18) and minimal change nephrotic syndrome (MCNS, n = 8), ATI with ischemia (n = 6), and drug-induced ATI (n = 7), were enrolled. ATI was evaluated by light microscopy. CD15 and IgG double staining and IgG subclass staining were performed to evaluate immunoglobulin deposition in the proximal tubular epithelium and TBM. IgG deposition was identified in the proximal tubules only in the FSGS group. Furthermore, IgG deposition in the TBM was observed in the FSGS group showing severe ATI. IgG3 was predominantly deposited by the IgG subclass study. Our results indicate that IgG deposition in the proximal tubular epithelium and TBM suggests the leaking of IgG from the glomerular filtration barrier and its reabsorption by proximal tubules, which may predict disruption of the glomerular size barrier, including subclinical FSGS. FSGS with ATI should be included as a differential diagnosis when IgG deposition in TBM is observed.

Human Parainfluenza Virus Type 2 V Protein Modulates Iron Homeostasis.

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.

Three-Dimensional Analysis of Interstitial Cells in the Smooth Muscle Layer of Murine Vas Deferens Using Confocal Laser Scanning Microscopy and FIB/SEM.

The smooth muscle contraction of the vas deferens has the important function of transporting sperm. Interstitial cells (ICs) play a critical role in the pacing and modulation of various smooth muscle organs by interactions with nerves and smooth muscle. Elucidating the three-dimensional (3D) architecture of ICs is important for understanding their spatial relationship on the mesoscale between ICs, smooth muscle cells (SMCs), and nerves. In this study, the 3D ultrastructure of ICs in the smooth muscle layer of murine vas deferens and the spatial relationships between ICs, nerves, and smooth muscles were observed using confocal laser scanning microscopy and focused ion beam/scanning electron microscopy. ICs have sheet-like structures as demonstrated by 3D observation using modern analytical techniques. Sheet-like ICs have two types of 3D structures, one flattened and the other curled. Multiple extracellular vesicle (EV)-like structures were frequently observed in ICs. Various spatial relations were observed in areas between ICs, nerves, and SMCs, which formed a complex 3D network with each other. These results suggest that ICs in the smooth muscle layer of murine vas deferens may have two subtypes with different sheet-like structures and may be involved in neuromuscular signal transmission via physical interaction and EVs.

The control of paramyxovirus genome hexamer length and mRNA editing.

The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NPQ202A) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NPwt is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln202 of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the cis-acting mRNA editing sequence is maintained.

A Minigenome Study of Hazara Nairovirus Genomic Promoters.

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3' and 5' strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

Common and unique mechanisms of filamentous actin formation by viruses of the genus Orthorubulavirus.

We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.

Three-dimensional ultrastructural analysis and histomorphometry of collagen bundles in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

BACKGROUND AND OBJECTIVE: The periodontal ligament (PDL) is an essential tissue for tooth function. However, the 3-dimensional ultrastructure of these PDL collagen bundles on a mesoscale is not clear. We investigated the 3-dimensional ultrastructure of these collagen bundles and quantitatively analyzed their histomorphometry using focused ion beam/scanning electron microscope (FIB/SEM) tomography. MATERIAL AND METHODS: The PDLs of the first mandibular molar of male C57BL/6 mice were analyzed using FIB/SEM tomography. The serial images of the collagen bundles so obtained were reconstructed. The collagen bundles were analyzed quantitatively using 3-dimensional histomorphometry. RESULTS: Collagen bundles of the PDL demonstrated multiple branched structures, rather than a single rope-like structure, and were wrapped in cytoplasm sheets. The structure of the horizontal fiber of the collagen bundle was an extensive meshwork. In contrast, the oblique and apical fibers of the collagen bundle showed a chain-like structure. The area and the minor and major axis lengths of cross-sections of the horizontal fiber, as determined from 3-dimensional images, were significantly different from those of the oblique and apical fibers. CONCLUSION: These findings indicate that collagen bundles in horizontal fiber areas have high strength and that the tooth is firmly anchored to the alveolar bone by the horizontal fibers, but is not secured evenly to the alveolar bone. The tooth is firmly anchored around the cervical area, creating a "slingshot-like structure." This study has provided further insights into the structure of the PDL and forms the basis for the development of more effective therapies for periodontal tissue regeneration.

Analysis of Scars and Keloids by Focused Ion Beam/Scanning Electron Microscopy: Distinguishing Between Hypertrophic Scars and Keloids.

BACKGROUND: Histological differentiation between hypertrophic scars (HSs) and keloids has been considered difficult. In this study, we analyzed differences in the 3-dimensional tissue architecture between HSs and keloids using focused ion beam/scanning electron microscopy (FIB/SEM). METHODS: Five specimens each of normal skin, normotrophic scars (NSs), HSs, and keloids were investigated. Three sites in each specimen were observed by FIB/SEM tomography, resulting in an observation of 15 sites per tissue type. We identified fibroblasts and macrophages and assessed the contact ratio and the mode of intercellular contact (planar contact or point contact). The significance of differences among the 4 tissue types was determined by Fisher exact test. RESULTS: In normal skin, contact between fibroblasts and macrophages was observed at all 15 sites, and the mode of contact was always planar. There was contact at 87% of the NS sites (planar: point = 80%: 7%). In HSs, contact was seen at 80% of the sites (planar: point = 20%: 60%). In keloids, contact was found at only 15% of the sites (planar: point = 7.5%: 7.5%). The intercellular contact ratio showed no significant differences among normal skin, NSs, and HSs; however, a significant difference was noted between these tissues and keloids. The intercellular contact mode also showed no significant difference between normal skin and NSs, but a significant difference between these tissues and HSs. CONCLUSIONS: These histopathologic findings suggest that FIB/SEM tomography is useful for distinguishing between HSs and keloids and can provide important knowledge for understanding the pathogenesis of keloids.

[A Case of Ileus Due to Colon Cancer with Non-Occlusive Mesenteric Ischemia].

Abdominal computed tomography(CT)revealed ileus due to sigmoid colon cancer in a 68-year-old man with abdominal pain, and endoscopic decompression using a transanal ileus tube was attempted. The blood test on the following day showed a marked increase in CRP 46.13mg/dL. Abdominal contrast CT was performed, and mesenteric ischemia was confirmed. Emergency surgery was performed on the same day. The ileum, and ascending, transverse, and descending colon appeared mottled and necrotic and were excised. A specialized diet started on the 5th postoperative day, and parenteral nutrition was used for a long period of time, due to the possibility of short bowel syndrome. The ileostomy and colostomy was closed 57 days after the operation. The patient finished parenteral nutrition on the 88th postoperative day without obvious nutritional absorption disorder and was discharged on the 94th postoperative day as oral intake only. We reported a case of ileus due to colon cancer with non-occlusive mesenteric ischemia(NOMI).

[A Case in Which the ICG Fluorography Was Useful for Detecting Minute Peritoneal Disseminations].

A 65-year-old man was emergently brought to our hospital because of rupture of 10 cm hepatocellular carcinoma(HCC) at left lobe in September 2019. He underwent selective transcatheter arterial embolization(TAE)for hemostasis. Enhanced computed tomography(CT)revealed one more 26 mm HCC at segment 8(S8)in addition to the ruptured HCC. Transcatheter arterial chemoembolization(TACE)was performed for both tumors. HCC at left lobe was resistant to TACE, hence we performed left hepatectomy. During the surgery we searched for peritoneal dissemination by using indocyanine green(ICG) fluorography and found 4 nodules with ICG accumulation in the omentum. All the nodules were pathologically diagnosed as peritoneal dissemination. We reported a case in which the ICG fluorography was very useful for detecting small peritoneal disseminations.

Importance of tyrosine in the RNA-binding domain of human parainfluenza virus type 2 nucleoprotein for polymerase activity.

The RNA genome of human parainfluenza virus type 2 (hPIV2) is encapsidated by nucleoprotein (NP) to act as a template for RNA synthesis. We examined the importance of individual amino acids in the RNA-binding domain of hPIV2 NP for polymerase activity using a mini-replicon assay. We showed that substitution of tyrosine at amino acid position 260, located in the RNA-binding pocket of NP, severely reduced polymerase activity. The aromatic side-chain of Y260 may be required for the formation of stable contacts between nucleotides and basic amino acids, thereby affecting promoter recognition by the viral polymerase.