Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Retrospective study on the possibility of an SFTS outbreak associated with undiagnosed febrile illness in veterinary professionals and a family with sick dogs in 2003.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-born disease and its animal-to-human transmission has come to attention recently. During our sero-survey of SFTS virus (SFTSV) among veterinary professionals in 2018, a veterinarian and his assistant working in an animal hospital were tested positive by enzyme-linked immunosorbent assay (ELISA). An additional survey implied a cluster of SFTS cases in which four more people, a family who brought two sick dogs to the animal hospital in 2003, were involved. This study aimed at assessing the possibility of animal-to-human transmission of SFTSV in this cluster. METHODS: Retrospective interviews were performed with the owner family of the dogs and their clinical records were obtained from each hospital. SFTSV-IgG were tested by ELISA and virus neutralization test using the sera collected from them in 2018. RESULTS: The interviews revealed that a total of six people, the two veterinary professionals and the owner family who took care of the sick dogs, suffered from SFTS-like symptoms in the same period of time in 2003. All patients did not have tick bite before the onset and all suspected causative agents were excluded by laboratory tests. The serological tests in this study revealed the four owner family members were all positive for SFTSV antibodies. CONCLUSIONS: Considering the extremely low seroprevalence of SFTSV antibodies among inhabitants of the region, the existence of SFTSV antibodies in all these six people presents a possibility that they were involved in an SFTS outbreak originated in the sick dogs in 2003.

Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury.

BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.

Development of a Dengue Virus Serotype-Specific Non-Structural Protein 1 Capture Immunochromatography Method.

Four serotypes of dengue virus (DENV), type 1 to 4 (DENV-1 to DENV-4), exhibit approximately 25-40% of the difference in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from specimens followed by PCR amplification is the current standard method of DENV serotype determination. However, since this method is time-consuming, rapid detection systems are desirable. We established several mouse monoclonal antibodies directed against DENV non-structural protein 1 and integrated them into rapid DENV detection systems. We successfully developed serotype-specific immunochromatography systems for all four DENV serotypes. Each system can detect 104 copies/mL in 15 min using laboratory and clinical isolates of DENV. No cross-reaction between DENV serotypes was observed in these DENV isolates. We also confirmed that there was no cross-reaction with chikungunya, Japanese encephalitis, Sindbis, and Zika viruses. Evaluation of these systems using serum from DENV-infected individuals indicated a serotype specificity of almost 100%. These assay systems could accelerate both DENV infection diagnosis and epidemiologic studies in DENV-endemic areas.

Direct Transmission of Severe Fever with Thrombocytopenia Syndrome Virus from Domestic Cat to Veterinary Personnel.

Two veterinary personnel in Japan were infected with severe fever with thrombocytopenia syndrome virus (SFTSV) while handling a sick cat. Whole-genome sequences of SFTSV isolated from the personnel and the cat were 100% identical. These results identified a nosocomial outbreak of SFTSV infection in an animal hospital without a tick as a vector.

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.

Hepatocellular carcinoma with intracranial metastasis in a Japanese macaque (Macaca fuscata).

BACKGROUND: A 23-year-old male Japanese macaque (Macaca fuscata) showed left ptosis, which progressed to exophthalmos. METHODS: The macaque underwent a clinical examination, CT and MRI, and was euthanized. Necropsy and histopathological examination were performed after euthanasia. RESULTS: The CT revealed and MRI confirmed an intracranial mass at the skull base with orbital extension. At necropsy, there were a large hepatic mass and an intracranial mass compressing the left temporal lobe of the brain. Histopathological and immunohistological examinations revealed that the masses were hepatocellular carcinoma (HCC) and a metastatic lesion. In both the primary and metastatic lesions, neoplastic hepatocytes were arranged mainly in a trabecular pattern. Immunohistochemically, the tumor cells were positive for cytokeratin (AE1/AE3 and CAM5.2) and hepatocyte paraffin 1 and negative for cytokeratin 7 and 20 and vimentin. CONCLUSION: To our knowledge, this is the first case report of HCC with intracranial metastasis in a macaque.

[A Case of Multiple Venous Thromboses Associated with Acute Cytomegalovirus Infection].

A previously healthy 44-year-old male presented with fever, abdominal pain, liver dysfunction and lymphadenopathy. He was diagnosed as having acute cytomegalovirus (CMV) infection with elevated CMV-IgG and IgM, and observed with supportive therapy. He was admitted to our hospital with prolonged fever lasting for a month. Enhanced CT revealed multiple thromboses in the right pulmonary artery and superior mesenteric vein. Follow-up CT after one week revealed new-onset thromboses in the left pulmonary artery and common iliac vein. Screening tests for thrombophilia were negative. His symptoms were improved with anticoagulant therapy with intravenous heparin, followed by oral warfarin. He was discharged on admission day 28 with good condition. Follow-up CT after 6 months revealed complete resolution of the thromboses. Anticoagulant therapy was stopped after 9 months, and he has been well without recurrence. Though vascular thrombosis is a rare complication, we must be alert to the signs and symptoms of thrombosis in patients with acute CMV infection.

Patterning of mammalian heterodont dentition within the upper and lower jaws.

Mammalian heterodont dentition is differentiated into incisors, canines, premolars, and molars in the mesial-distal direction, in both the upper and lower jaws. Although all the lower teeth are rooted in the mandible, the upper incisors are rooted in the premaxilla and the upper canine and the teeth behind it are in the maxilla. The present study uncovers ontogenetic backgrounds to these shared and differing mesiodistal patterns of the upper and lower dentition. To this end, we examined the dentition development of the house shrew, Suncus murinus, instead of the rodent model animals because the dentition of this primitive eutherian species includes all the tooth classes, and no toothless diastema region. In the shrew, the upper incisor-forming region extended over the medial nasal prominence and the mesial part of the maxillary prominence. Consequently, the maxillary and mandibular prominences were in a mirror-image relationship in terms of the mesiodistally differentiated tooth-forming regions and of the complementary gene expression pattern, with Bmp4 in the mesial and Fgf8 in the distal regions. This suggests shared molecular mechanisms regulating tooth class differentiation between the upper and lower jaws. However, the premaxillary bone appeared within the mesenchyme of the medial nasal prominence, but grew distally beyond the former epithelial boundary with the maxillary prominence to form, finally, the incisive (premaxillary-maxillary) suture just mesial to the canine. Therefore, the developmental locations of the upper incisors are not inconsistent with the classical osteological criterion of the upper canine by comparative odontologists.

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

[Streptococcal Toxic Shock-like Syndrome due to Streptococcus suis Serotype 2 in a Japanese Pig Farmer].

Streptococcus suis is a major swine pathogen. It has recently been recognized as an emerging zoonosis that causes mainly meningitis and sepsis in human. In particular, toxic shock-like syndrome (TSLS) caused by this pathogen has a high mortality rate. However, misidentification of S. suis by conventional biochemical and commercial identification tests is not rare. The patient was a 71-year-old man who worked as a pig farmer who was admitted for fever, oliguria and subcutaneous hemorrhage. He was diagnosed as having septic shock and blood culture was positive for Gram-positive cocci, mainly diplococcus. This pathogen was identified with S. suis with using MALDI-TOF MS analysis, though a commercial Gram-Positive bacteria identification kit revealed viridans streptococci. His clinical features met the diagnostic criteria of TSLS, and ceftriaxone and clindamycin were administered. On admission day 28, he was discharged in good condition.

A mouse monoclonal antibody against dengue virus type 1 Mochizuki strain targeting envelope protein domain II and displaying strongly neutralizing but not enhancing activity.

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.

Developmental process of the modern house shrew's molars: implications for the evolution of the tribosphenic molar in Mesozoic mammals.

Phylogenetically, the tribosphenic molars-prototypes of multi-cusped cheek teeth in marsupial and placental mammals-are derived from the single-cusped conical teeth of reptiles through the addition of cusps. Ontogenetically, mammalian molars are formed through the interface between the dental epithelium and mesenchyme (future enamel-dentin junction), becoming geometrically complex by adding epithelial signaling centers, called enamel knots, which determine future cusp positions. To reevaluate cusp homologies in Mesozoic mammals from an ontogenetic perspective, this study tracked molar development in a living placental mammal species, the house shrew (Suncus murinus), whose molars are morphologically the least derived from tribosphenic prototypes. The development of shrew molars proceeded as if it replayed the evolutionary process of tribosphenic molars. The first formed enamel knots gave rise to the evolutionarily oldest cusps-upper paracone and lower protoconid. The order of formation of other enamel knots and their location in development seemed to trace the order of cusp appearance in evolution. The parallel relationship between ontogeny and phylogeny of mammalian molars, if any, suggests that a change in the timing between developmental events rather than a change in the morphogenetic mechanism itself, should have been a major causal factor for the evolutionary transformation of tooth morphology.

Three-dimensional topography of rat trigeminal ganglion neurons using a combination of retrograde labeling and tissue-clearing techniques.

The trigeminal nerve is the sensory afferent of the orofacial regions and divided into three major branches. Cell bodies of the trigeminal nerve lie in the trigeminal ganglion and are surrounded by satellite cells. There is a close interaction between ganglion cells via satellite cells, but the function is not fully understood. In the present study, we clarified the ganglion cells' three-dimensional (3D) localization, which is essential to understand the functions of cell-cell interactions in the trigeminal ganglion. Fast blue was injected into 12 sites of the rat orofacial regions, and ganglion cells were retrogradely labeled. The labeled trigeminal ganglia were cleared by modified 3DISCO, imaged with confocal laser-scanning microscopy, and reconstructed in 3D. Histograms of the major axes of the fast blue-positive somata revealed that the peak major axes of the cells innervating the skin/mucosa were smaller than those of cells innervating the deep structures. Ganglion cells innervating the ophthalmic, maxillary, and mandibular divisions were distributed in the anterodorsal, central, and posterolateral portions of the trigeminal ganglion, respectively, with considerable overlap in the border region. The intermingling in the distribution of ganglion cells within each division was also high, in particular, within the mandibular division. Specifically, intermingling was observed in combinations of tongue and masseter/temporal muscles, maxillary/mandibular molars and masseter/temporal muscles, and tongue and mandibular molars. Double retrograde labeling confirmed that some ganglion cells innervating these combinations were closely apposed. Our data provide essential information for understanding the function of ganglion cell-cell interactions via satellite cells.

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

Development of a rapid assay system for detecting antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is one of the pathogenic mechanisms related to disease severity in dengue virus infection. Conventional assays for detecting ADE activity usually require several days. In this study, we established a rapid assay system to evaluate ADE activity in dengue-seropositive samples using single round infectious particles (SRIPs). Human Fc-gamma receptor-bearing cells (K562 and Mylc cells) were infected with SRIP antigen in the presence of human serum samples to measure ADE activity. Two assay protocols were introduced: (i) rapid assay with 5 h of incubation, and (ii) semi-rapid assay with 24 h of incubation. The rapid assay requires a large quantity of SRIP antigen and gives results in half a day. Although the semi-rapid assay requires slightly more than a day, it can be performed using only a small amount of SRIP. Interestingly, the range of the number of Mylc cells required for the semi-rapid assay was wider than that of K562 cells. Significant correlations were observed between the rapid and semi-rapid assays for both cell types. Although it is difficult to judge which protocol best reflects the current immune status in vivo, both assays could rapidly provide valuable information regarding the risk assessment for severe diseases.

Potential Protective Effect of Dengue NS1 Human Monoclonal Antibodies against Dengue and Zika Virus Infections.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1ß, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.

Identification of the Tembusu Virus in Mosquitoes in Northern Thailand.

Among emerging zoonotic pathogens, mosquito-borne viruses (MBVs) circulate between vertebrate animals and mosquitoes and represent a serious threat to humans via spillover from enzootic cycles to the human community. Active surveillance of MBVs in their vectors is therefore essential to better understand and prevent spillover and emergence, especially at the human-animal interface. In this study, we assessed the presence of MBVs using molecular and phylogenetic methods in mosquitoes collected along an ecological gradient ranging from rural urbanized areas to highland forest areas in northern Thailand. We have detected the presence of insect specific flaviviruses in our samples, and the presence of the emerging zoonotic Tembusu virus (TMUV). Reported for the first time in 1955 in Malaysia, TMUV remained for a long time in the shadow of other flaviviruses such as dengue virus or the Japanese encephalitis virus. In this study, we identified two new TMUV strains belonging to cluster 3, which seems to be endemic in rural areas of Thailand and highlighted the genetic specificities of this Thai cluster. Our results show the active circulation of this emerging flavivirus in Thailand and the need for continuous investigation on this poorly known but threatening virus in Asia.

Resistance of Aedes aegypti (L.) larvae to temephos in Surabaya, Indonesia.

The resistance of Aedes aegypti mosquitoes to insecticides threatens dengue virus control efforts. In this study, Ae. aegypti larvae collected from 12 subdistricts in Surabaya, Indonesia, where dengue is endemic, were tested for resistance to the organophosphate, temephos. Susceptibility testing, performed according to World Health Organization (WHO) methods, showed all field strains were resistant to temephos at a dose of 0.012 mg/l, with mortality rates at 24 hours of 22% to 60%. Another susceptibility test to determine median lethal time (LT50) indicates resistance ratios ranging from 2.2 to 8.5. Although incipient resistance was detected at a dosage of 1 mg/l, as determined by the LT50, mortalities higher than 80% within 24 hours were detected using the WHO method in nine subdistricts of Surabaya, indicating temephos at 1 mg/l is still effective in field conditions in these areas. In three subdistricts (Tambaksari, Gubeng and Sawahan), the mortality rates were under 80%, indicating possible resistance to temephos.