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Background: Hemostasis is very important for a safe surgery, particularly in endoscopic surgery. Accordingly, in the last decade, vessel-sealing systems became popular as hemostatic devices. However, their use is limited due to thermal damage to organs, such as intestines and nerves. We developed a new method for safe coagulation using a vessel-sealing system, termed flat coagulation (FC). This study aimed to evaluate the efficacy of this new FC method compared to conventional coagulation methods. Methods: We evaluated the thermal damage caused by various energy devices, such as the vessel-sealing system (FC method using LigaSure™), ultrasonic scissors (Sonicision™), and monopolar electrosurgery (cut/coagulation/spray/soft coagulation (SC) mode), on porcine organs, including the small intestine and liver. Furthermore, we compared the hemostasis time between the FC method and conventional methods in the superficial bleeding model using porcine mesentery. Results: FC caused less thermal damage than monopolar electrosurgery's SC mode in the porcine liver and small intestine (liver: mean depth of thermal damage, 1.91 ± 0.35 vs 3.37 ± 0.28 mm; p = 0.0015). In the superficial bleeding model, the hemostasis time of FC was significantly shorter than that of electrosurgery's SC mode (mean, 19.54 ± 22.51 s vs 44.99 ± 21.18 s; p = 0.0046). Conclusion: This study showed that the FC method caused less thermal damage to porcine small intestine and liver than conventional methods. This FC method could provide easier and faster coagulation of superficial bleeds compared to that achieved by electrosurgery's SC mode. Therefore, this study motivates for the use of this new method to achieve hemostasis with various types of bleeds involving internal organs during endoscopic surgeries. [ABSTRACT FROM AUTHOR]

Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine. Author summary: Human herpesvirus 6B (HHV-6B) is known as the cause of the common childhood febrile illness exanthem subitum in its primary infection, and it develops into a lifelong latent infection in almost all individuals. Severe complications such as meningitis and encephalitis can occur in both the primary infection and reactivation. There is no established treatment or vaccine. The tetrameric glycoprotein complex gH/gL/gQ1/gQ2 (tetramer) on the viral envelope is the ligand for the entry of HHV-6B, which is the critical part for its infection. Here, we established a soluble form of the tetramer and purified it to homogeneity. After several immunizations of tetramer along with different combinations of adjuvants in mice, we observed that it greatly induced defensive immunity against HHV-6B, indicating that the tetramer has the potential to become a vaccine candidate. Moreover, our results also revealed that combinations of distinct adjuvants with the tetramer would be useful as an HHV-6B vaccine strategy for different purposes. [ABSTRACT FROM AUTHOR]

A unique glycoprotein is expressed on the virus envelope of human herpesvirus 6B (HHV-6B): the complex gH/gL/gQ1/gQ2 (hereafter referred to as the HHV-6B tetramer). This tetramer recognizes a host receptor expressed on activated T cells: human CD134 (hCD134). This interaction is essential for HHV-6B entry into the susceptible cells and is a determinant for HHV-6B cell tropism. The structural mechanisms underlying this unique interaction were unknown. Herein we solved the interactions between the HHV-6B tetramer and the receptor by using their neutralizing antibodies in molecular and structural analyses. A surface plasmon resonance analysis revealed fast dissociation/association between the tetramer and hCD134, although the affinity was high (KD = 18 nM) and comparable to those for the neutralizing antibodies (anti-gQ1: 17 nM, anti-gH: 2.7 nM). A competition assay demonstrated that the anti-gQ1 antibody competed with hCD134 in the HHV-6B tetramer binding whereas the anti-gH antibody did not, indicating the direct interaction of gQ1 and hCD134. A single-particle analysis by negative-staining electron microscopy revealed the tetramer's elongated shape with a gH/gL part and extra density corresponding to gQ1/gQ2. The anti-gQ1 antibody bound to the tip of the extra density, and anti-gH antibody bound to the putative gH/gL part. These results highlight the interaction of gQ1/gQ2 in the HHV-6B tetramer with hCD134, and they demonstrate common features among viral ligands of the betaherpesvirus subfamily from a macroscopic viewpoint. Author summary: Primary infection of human herpesvirus 6B (HHV-6B) with fever and roseola occurs for almost all children, and HHV-6B remains in the host as a latent infection. The reactivation of HHV-6B (especially in patients after hematopoietic stem-cell transplantation) occasionally causes severe encephalitis, which is a public health concern worldwide. HHV-6B's unique gH/gL/gQ1/gQ2 complex (a tetramer expressed on the viral envelope) recognizes the cellular receptor, i.e., human CD134 (hCD134), which is essential for the entry of the virus into the host's cells. The interaction between HHV-6B tetramer and hCD134 is therefore one of the determinants of HHV-6B-specific cell tropism. This article sheds light on the molecular and structural interactions between HHV-6B tetramer and its host receptor along with their neutralizing antibodies, and their affinities, competition, and binding modes based on the HHV-6B tetramer structure are described. Our findings provide molecular and structural bases for a comprehensive understanding of these interactions and relationships. [ABSTRACT FROM AUTHOR]

Purpose: Imaging findings of pilocytic astrocytoma (PA) vary widely, sometimes resembling those of high-grade glioma (HGG). This study aimed to identify the imaging parameters that can be used to differentiate PA from HGG.Methods: Altogether, 60 patients with PAs and 138 patients with HGGs were included in the study. Tumor properties and the presence of hydrocephalus, peritumoral edema, and dissemination were evaluated. We also measured the maximum relative cerebral blood flow (rCBFmax) and volume (rCBVmax) and determined the minimum apparent diffusion coefficient (ADCmin) in the tumor’s solid components. The relative T1 (rT1), T2 (rT2), and contrast-enhanced T1 (rCE-T1) intensity values were evaluated. Parameters were compared between PAs and HGGs using the Mann-Whitney U test. Receiver operating characteristic (ROC) curve analysis was also used to evaluate these imaging parameters. A value of P < .05 was considered to indicate significance.Results: Intratumoral hemorrhage and calcification were observed in 10.0% and 21.7% of PAs, respectively. The rCBFmax and rCBVmax values were significantly lower in PAs (0.50 ± 0.35, 1.82 ± 1.21) than those in HGGs (2.98 ± 1.80, 9.54 ± 6.88) (P < .0001, P = .0002, respectively). The ADCmin values were significantly higher in PAs (1.36 ± 0.56 × 10−3 mm2/s) than those in HGGs (0.86 ± 0.37 × 10−3 mm2/s) (P < .0001). ROC analysis showed that the best diagnostic performance was achieved with rCBFmax.Conclusion: The rCBFmax, rCBVmax, and ADCmin can differentiate PAs from HGGs. [ABSTRACT FROM AUTHOR]

Sensitivity and/or specificity of the in vivo erythrocyte micronucleus (MN) and transgenic rodent mutation (TGR) tests to detect rodent carcinogens and non-carcinogens were investigated. The Carcinogenicity and Genotoxicity eXperience (CGX) dataset created by Kirkland et al. was used for the carcinogenicity and in vitro genotoxicity data, i.e. , Ames and chromosome aberration (CA) tests. Broad literature surveys were conducted to gather in vivo MN or TGR test data to add to the CGX dataset. Genotoxicity data in vitro were also updated slightly. Data on 379 chemicals (293 carcinogens and 86 non-carcinogens) were available for the in vivo MN test; sensitivity, specificity or concordances were calculated as 41.0%, 60.5% or 45.4%, respectively. For the TGR test, data on 80 chemicals (76 carcinogens and 4 non-carcinogens) were available; sensitivity was calculated as 72.4%. Based on the recent guidance on genotoxicity testing strategies, performance (sensitivity/specificity) of the following combinations was calculated; Ames + in vivo MN (68.7%/45.3%), Ames + TGR (83.8%/not calculated (nc)), Ames + in vitro CA + in vivo MN (80.8%/21.3%), Ames + in vitro CA + TGR (89.1%/nc), Ames + in vivo MN + TGR (87.5%/nc), Ames + in vitro CA + in vivo MN + TGR (89.3%/nc). Relatively good balance in performance was shown by the Ames + in vivo MN in comparison with Ames + in vitro CA (74.3%/37.5%). Ames + TGR and Ames + in vivo MN + TGR gave even higher sensitivity, but the specificity could not be calculated (too few TGR data on non-carcinogens). This indicates that in vivo MN and TGR tests are both useful as in vivo tests to detect rodent carcinogens. [ABSTRACT FROM AUTHOR]

Straining of a horizontal density gradient by tidal currents acts to periodically produce and destroy near-bottom stratification, which has been shown to modulate turbulence in the bottom boundary layer (BBL). Previous observations of such periodic variations have been limited to the coastal ocean and estuaries, where horizontal density gradients are maintained by river runoff or differential heating. In the present study, we show evidence for the existence of tidal straining over the continental shelf, outside any regions of freshwater influence, where horizontal density gradients are likely to result from the projection of the interior vertical stratification onto sloping topography. Based on microstructure data obtained in the East China Sea, we demonstrate that the tidal current shear interacting with the cross-isobath density gradient results in semidiurnal switching between unstable and stable stratification in the lower part of the BBL. The cycle of turbulent dissipation is quarter-diurnal, corresponding to the semidiurnal variation of tidal current shear. In addition, a noticeable diurnal modulation in stratification as well as a significant diurnal cycle of turbulent dissipation are observed in the upper part of the BBL, where the time evolution of stratification is dominated by tidal advection, rather than tidal straining. [ABSTRACT FROM AUTHOR]

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans -Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. [ABSTRACT FROM AUTHOR]

All herpesviruses contain a tegument layer comprising a protein matrix; these proteins play key roles during viral assembly and egress. Here, liquid chromatography and tandem mass spectrometry analysis (LC–MS/MS) of proteins from human herpesvirus 6 (HHV-6)-infected cells revealed a possible association between two major tegument proteins, U14 and U11. This association was verified by immunoprecipitation experiments. Moreover, U11 protein was expressed during the late phase of infection and incorporated into virions. Finally, in contrast to its revertant, a U11 deletion mutant could not be reconstituted. Taken together, these results suggest that HHV-6 U11 is an essential gene for virus growth and propagation. [ABSTRACT FROM AUTHOR]

We studied the distribution of implanted Xe in amorphous SiO 2 . Our results clearly showed that Xe profile is energy and fluence dependent. By varying ion energy from 50 to 300 keV, we found that its thermal out-diffusion is very conventional for the first two energies and unexpected for the highest energy. In that last case Xe main peak increases with thermal annealing. Instead of out-diffusing, Xe seems to be driven toward the main peak. The effect of ion fluence is similar to energy one with a conventional out-diffusion for lower fluences (5 × 10 15 and 1 × 10 16 cm −2 ) while higher fluences (3.5 × 10 16 and 5 × 10 16 cm −2 ) display an increase of the main peak with annealing. Such a behavior can be linked to the formation (or not) of a high density of stable bubbles. [ABSTRACT FROM AUTHOR]

Autosomal dominant leukodystrophy is an extremely rare disease. Here we report on a dominantly inherited disease in a Japanese family with slowly progressive clinical course. Their symptoms and signs started in early childhood and very slowly progressed. In most patients spastic gait was the initial symptom. Neurological manifestations were characterized by pyramidal signs, ataxia, and mental deterioration. In addition to these neurological signs, the skeletal anomalies such as scoliosis and congenital hip dislocation were also present. MR images showed no abnormality in the early stage, but T2-weighted images revealed high intensity areas in the cerebral and cerebellar white matter, and the dentate nucleus in the advanced stage. Proton MR spectroscopy showed decrease of N-acetylaspartate/creatine ratio and increase of choline/creatine ratio in the advanced stage. Proton MR spectroscopy revealed normal N-acetylaspartate/creatine ratio and increase of choline/creatine ratio in the early stage. We suggested that these patients had abnormality in the white matter when MRI was still normal. We considered that intracranial demyelination was gradually progressed as the symptoms got aggravated. [Copyright &y& Elsevier]

The reverse genetics system is a very powerful tool for analyzing the molecular mechanisms of viral propagation and pathogenesis. However, full-length genome plasmid construction is highly time-consuming and laborious, and undesired mutations may be introduced by Escherichia coli. This study shows a very rapid E. coli-free method of full-genome construction using the mumps virus as an example. This method was able to reduce dramatically the time for full-genome construction, which was used very efficiently for virus rescue, from several days or more to ~2 days, with a similar accuracy and yield to the conventional method using E. coli/plasmid., (© 2022 The Authors. Microbiology and Immunology published by The Societies and John Wiley & Sons Australia, Ltd.)

The nucleolus is the largest structure in the nucleus, and it plays roles in mediating cellular stress responses and regulating cell proliferation, as well as in ribosome biosynthesis. The nucleolus is composed of a variety of nucleolar factors that interact with each other in a complex manner to enable its function. Many viral proteins interact with nucleolar factors as well, affecting cellular morphology and function. Here, to investigate the association between mumps virus (MuV) infection and the nucleolus, we evaluated the necessity of nucleolar factors for MuV proliferation by performing a knockdown of these factors with small interfering (si)RNAs. Our results reveal that suppressing the expression of Treacle, which is required for ribosome biosynthesis, reduced the proliferative potential of MuV. Additionally, the one-step growth kinetics results indicate that Treacle knockdown did not affect the viral RNA and protein synthesis of MuV, but it did impair the production of infectious virus particles. Viral matrix protein (M) was considered a candidate Treacle interaction partner because it functions in the process of particle formation in the viral life cycle and is partially localized in the nucleolus. Our data confirm that MuV M can interact with Treacle and colocalize with it in the nucleolus. Furthermore, we found that viral infection induces relocalization of Treacle in the nucleus. Together, these findings suggest that interaction with Treacle in the nucleolus is important for the M protein to exert its functions late in the MuV life cycle. IMPORTANCE The nucleolus, which is the site of ribosome biosynthesis, is a target organelle for many viruses. It is increasingly evident that viruses can favor their own replication and multiplication by interacting with various nucleolar factors. In this study, we found that the nucleolar protein Treacle, known to function in the transcription and processing of pre-rRNA, is required for the efficient propagation of mumps virus (MuV). Specifically, our data indicate that Treacle is not involved in viral RNA or protein synthesis but is important in the processes leading to viral particle production in MuV infection. Additionally, we determined that MuV matrix protein (M), which functions mainly in viral particle assembly and budding, colocalized and interacted with Treacle. Furthermore, we found that Treacle is distributed throughout the nucleus in MuV-infected cells. Our research shows that the interaction between M and Treacle supports efficient viral growth in the late stage of MuV infection.

Many efforts have been dedicated to the discovery of antiviral drug candidates against the mumps virus (MuV); however, no specific drug has yet been approved. The development of efficient screening methods is a key factor for the discovery of antiviral candidates. In this study, we evaluated a screening method using an Aequorea coerulescens green fluorescent protein-expressing MuV infectious molecular clone. The application of this system to screen for active compounds against MuV replication revealed that CD437, a retinoid acid receptor agonist, has anti-MuV activity. The point of antiviral action was a late step(s) in the MuV life cycle. The replication of other paramyxoviruses was also inhibited by CD437. The induction of retinoic acid-inducible gene (RIG)-I expression is a reported mechanism for the antiviral activity of retinoids, but our results indicated that CD437 did not stimulate RIG-I expression. Indeed, we observed antiviral activity despite the absence of RIG-I, suggesting that CD437 antiviral activity does not require RIG-I induction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kato, Nakatsu, Murano, Wakata, Kubota, Hishiki, Yamaji, Kidokoro, Katoh and Takeda.)

Human herpesvirus 6A (HHV-6A) and HHV-6B use different cellular receptors, human CD46 and CD134, respectively and have different cell tropisms although they have 90% similarity at the nucleotide level. An important feature that characterizes HHV-6A/6B is the glycoprotein H (gH)/gL/gQ1/gQ2 complex (a tetramer) that each virus has specifically on its envelope. Here, to determine which molecules in the tetramer contribute to the specificity for each receptor, we developed a cell-cell fusion assay system for HHV-6A and HHV-6B that uses the cells expressing CD46 or CD134. With this system, when we replaced the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion activity mediated by glycoproteins via CD46 was lower than that done with the original-type tetramer. When we replaced the gQ1 or the gQ2 of HHV-6A with that of HHV-6B in the tetramer, the cell fusion mediated by glycoproteins via CD134 was not seen. In addition, we generated two types of C-terminal truncation mutants of HHV-6A gQ2 (AgQ2) to examine the interaction domains of HHV-6A gQ1 (AgQ1) and AgQ2. We found that amino acid residues 163 to 185 in AgQ2 are important for interaction of AgQ1 and AgQ2. Finally, to investigate whether HHV-6B gQ2 (BgQ2) can complement AgQ2, an HHV-6A genome harboring BgQ2 was constructed. The mutant could not produce an infectious virus, indicating that BgQ2 cannot work for the propagation of HHV-6A. These results suggest that gQ2 supports the tetramer's function, and the combination of gQ1 and gQ2 is critical for virus propagation. IMPORTANCE Glycoprotein Q2 (gQ2), an essential gene for virus propagation, forms a heterodimer with gQ1. The gQ1/gQ2 complex has a critical role in receptor recognition in the gH/gL/gQ1/gQ2 complex (a tetramer). We investigated whether gQ2 regulates the specific interaction between the HHV-6A or -6B tetramer and CD46 or CD134. We established a cell-cell fusion assay system for HHV-6A/6B and switched the gQ1 or gQ2 of HHV-6A with that of HHV-6B in the tetramer. Although cell fusion was induced via CD46 when gQ1 or gQ2 was switched between HHV-6A and -6B, the activity was lower than that of the original combination. When gQ1 or gQ2 was switched in HHV-6A and -6B, no cell fusion was observed via CD134. HHV-6B gQ2 could not complement the function of HHV-6A's gQ2 in HHV-6A propagation, suggesting that the combination of gQ1 and gQ2 is critical to regulate the specificity of the tetramer's function for virus entry., (Copyright © 2020 American Society for Microbiology.)

Human herpesvirus 6A (HHV-6A) is a member of the genus Roseolovirus and the subfamily Betaherpesvirinae. It is similar to and human cytomegalovirus (HCMV). HHV-6A encodes a 41 kDa nuclear phosphoprotein, U27, which acts as a processivity factor in the replication of the viral DNA. HHV-6A U27 has 43% amino acid sequence homology with HCMV UL44, which is important for DNA replication. A previous study on HHV-6A U27 revealed that it greatly increases the in vitro DNA synthesis activity of HHV-6A DNA polymerase. However, the role of U27 during the HHV-6A virus replication process remains unclear. In this study, we constructed a U27-deficient HHV-6A mutant (HHV-6ABACU27mut) with a frameshift insertion at the U27 gene using an HHV-6A bacterial artificial chromosome (BAC) system. Viral reconstitution from the mutant BAC DNA was not detected, in contrast to the wild type and the revertant from the U27 mutant. This suggests that U27 plays a critical role in the life cycle of HHV-6A., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

Human herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity. IMPORTANCE Glycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents, suggesting that it might be a target of the immune system., (Copyright © 2018 American Society for Microbiology.)

Immediate early proteins of human herpesvirus 6A (HHV-6A) are expressed at the outset of lytic infection and thereby regulate viral gene expression. Immediate early protein 2 (IE2) of HHV-6A is a transactivator that drives a variety of promoters. The C-terminal region of HHV-6A IE2 is shared among IE2 homologs in betaherpesviruses and is involved in dimerization, DNA binding, and transcription factor binding. In this study, the structure of the IE2 C-terminal domain (IE2-CTD) was determined by X-ray crystallography at a resolution of 2.5 Å. IE2-CTD forms a homodimer stabilized by a β-barrel core with two interchanging long loops. Unexpectedly, the core structure resembles those of the gammaherpesvirus factors EBNA1 of Epstein-Barr virus and LANA of Kaposi sarcoma-associated herpesvirus, but the interchanging loops are longer in IE2-CTD and form helix-turn-helix (HTH)-like motifs at their tips. The HTH and surrounding α-helices form a structural feature specific to the IE2 group. The apparent DNA-binding site (based on structural similarity with EBNA1 and LANA) resides on the opposite side of the HTH-like motifs, surrounded by positive electrostatic potential. Mapping analysis of conserved residues on the three-dimensional structure delineated a potential factor-binding site adjacent to the expected DNA-binding site. The predicted bi- or tripartite functional sites indicate a role for IE2-CTD as an adapter connecting the promoter and transcriptional factors that drive gene expression. IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B belong to betaherpesvirus subfamily. Both viruses establish lifelong latency after primary infection, and their reactivation poses a significant risk to immunocompromised patients. Immediate early protein 2 (IE2) of HHV-6A and HHV-6B is a transactivator that triggers viral replication and contains a DNA-binding domain shared with other betaherpesviruses such as human herpesvirus 7 and human cytomegalovirus. In this study, an atomic structure of the DNA-binding domain of HHV-6A IE2 was determined and analyzed, enabling a structure-based understanding of the functions of IE2, specifically DNA recognition and interaction with transcription factors. Unexpectedly, the dimeric core resembles the DNA-binding domain of transcription regulators from gammaherpesviruses, showing structural conservation as a DNA-binding domain but with its own unique structural features. These findings facilitate further characterization of this key viral transactivator., (Copyright © 2017 American Society for Microbiology.)

Human herpesvirus 6A (HHV-6A) starts its replication cycle following the action of immediate early proteins that transactivate viral promoters. Immediate early protein 2 (IE2) of HHV-6A is a 1500 amino acid polypeptide with a C-terminal region that is conserved among beta-herpesvirus subfamily members. In this study, a structural domain in the homologous C-terminal region was subjected to secondary structure prediction, and residues 1324-1500 were subsequently designated as the C-terminal domain of IE2 (IE2-CTD). The gene fragment encoding IE2-CTD was inserted into an E. coli expression vector and expressed as a fusion protein with maltose binding protein (MBP) at the N-terminus. IE2-CTD has a theoretical isoelectric point (pI) of 9.29, and strong cation exchange column chromatography was effective for purification. Needle-shaped crystals of IE2-CTD were obtained using the sitting-drop vapour diffusion method, and larger selenomethionine-labelled crystals of space group P2₁ diffracted X-rays to 2.5 Å resolution using synchrotron radiation. Data were collected at the selenium absorption peak wavelength for experimental phasing by the single anomalous dispersion method. The resulting electron density map clearly shows the protein backbone, and full structural determination and refinement are in progress.

Membrane fusion, which is the key process for both initial cell entry and subsequent lateral spread of herpes simplex virus (HSV), requires the four envelope glycoproteins gB, gD, gH, and gL. Syncytial mutations, predominantly mapped to the gB and gK genes, confer hyperfusogenicity on HSV and cause multinucleated giant cells, termed syncytia. Here we asked whether interaction of gD with a cognate entry receptor remains indispensable for initiating membrane fusion of syncytial strains. To address this question, we took advantage of mutant viruses whose viral entry into cells relies on the uniquely specific interaction of an engineered gD with epidermal growth factor receptor (EGFR). We introduced selected syncytial mutations into gB and/or gK of the EGFR-retargeted HSV and found that these mutations, especially when combined, enabled formation of extensive syncytia by human cancer cell lines that express the target receptor; these syncytia were substantially larger than the plaques formed by the parental retargeted HSV strain. We assessed the EGFR dependence of entry and spread separately by using direct entry and infectious center assays, respectively, and we found that the syncytial mutations did not override the receptor specificity of the retargeted viruses at either stage. We discuss the implications of these results for the development of more effective targeted oncolytic HSV vectors., Importance: Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A new mechanistic principle for reporting the phosphorylation of tyrosine is described, which should prove applicable to even the most fastidious of protein tyrosine kinases, as demonstrated by the acquisition of a fluorescent sensor for the extraordinarily demanding anaplastic lymphoma kinase.

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.

The induction of micronuclei by methotrexate (MTX) was examined in two laboratories using mouse peripheral blood reticulocytes. MTX was a weak inducer in the micronucleus test using bone marrow cells and single treatments, and was one of the few chemicals showing a multiple-treatment effect (CSGMT/JEMS.MMS, 1990). In our preliminary experiments, the ratio of reticulocytes to total erythrocytes decreased greatly after a single treatment with MTX at 100 mg/kg, so lower dose levels of MTX were selected to carry out the micronucleus test in peripheral blood. Full-scale tests were performed at dose levels of 0, 10, 20, 40, and 80 mg/kg, with five sampling times of 0, 24, 48, 72, and 96 h. Frequencies of micronucleated reticulocytes (MNRETs) increased dose-dependently at 72 h, to a maximum of approximately 1%; some preparations obtained from the animals at higher doses could not be examined because the ratio of reticulocytes to total erythrocytes had decreased severely. At doses of 0.5-4.0 mg/kg, the effect of multiple treatments vs. single treatments was not clear, nor was the maximum level of response much different. Since MTX induced a clear positive response in peripheral blood reticulocytes after a single treatment, the reticulocytes in peripheral blood seem a more sensitive target.

3-Hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) is a direct-acting mutagenic compound derived by metabolic activation from 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), a strongly mutagenic carcinogen. The action of N-OH-Trp-P-2 on DNA in vitro was investigated. N-OH-Trp-P-2 inactivated Bacillus subtilis transforming DNA and produced single-strand cuts in a supercoiled circular DNA (phi X174RFI) under neutral conditions. When mouse FM3A cells in culture were treated with a noncytotoxic dose of N-OH-Trp-P-2 and then the cellular DNA was examined by the alkaline elution technique, chain cleavages of the DNA were observed. Cysteamine inhibited the spontaneous degradation of N-OH-Trp-P-2 and enhanced the covalent binding of [3H]N-OH-Trp-P-2 to DNA. This finding offered an explanation for the previously observed enhancement of Trp-P-2 mutagenicity by cysteamine. In contrast cysteamine inhibited the N-OH-Trp-P-2-mediated inactivation of B. subtilis DNA as well as the strand cleavage in phi X174RFI DNA. The cleavage in phi X174RFI DNA was also inhibited by catalase. These observations indicate that the mutagenicity and DNA-cleaving activity of N-OH-Trp-P-2 are distinct from each other, that the inactivation of transforming DNA was caused mainly by strand cleavage, and that the DNA cleavage was probably caused by active oxygen radicals produced in the oxidative degradation of N-OH-Trp-P-2.