Takako Suzuki

Puromycin aminonucleoside induces apoptosis and increases HNE in cultured glomerular epithelial cells

Abstract Puromycin aminonucleoside induces apoptosis and increases 4-hydroxy-2-nonenal (HNE) in cultured glomerular epithelial cells. We have previously reported the detachment of cultured glomerular epithelial cells (GECs) from their substrata by puromycin aminonucleoside (PAN) treatment. In this study we explored whether or not apoptosis was involved in the mechanisms of the detachment. DNA fragmentation on gel electrophoresis was clearly shown by 10 −3 M PAN treatment of GECs. Nuclear staining by Hoechst 33342 indicated the greatest number of apoptotic cells at 10 −3 M PAN for 48 h treatment. Similarly, TUNEL methods revealed maximal apoptotic cells at 10 −3 M PAN for 48 h treatment. Caspase-3 (like) protease activity increased at 10 −3 M PAN, and decreased at 2 × 10 −3 M PAN for 48 h treatment as well as at 10 −3 M PAN for 60 h treatment. Pretreatment with 2′-deoxycoformycin (DCF), inhibitor of adenosine deaminase, abolished these effects of PAN on cultured GECs. PAN treatment increased HNE, a lipid peroxide adduct, modified protein in cultured GECs, which was also prevented by pretreatment by DCF. These results for the first time indicate that the PAN-induced detachment of GECs from culture substrata is mediated at least in part through apoptosis via oxidative stresses by adenosine deaminase activity.

Coexpression of auxiliary Kvβ2 subunits with Kv1.1 channels is required for developmental acquisition of unique firing properties of zebrafish Mauthner cells

Each neuron possesses a unique firing property, which is largely attributed to heterogeneity in the composition of voltage-gated ion channel complexes. Zebrafish Mauthner (M) cells, which are bilaterally paired giant reticulospinal neurons (RSNs) in the hindbrain and induce rapid escape behavior, generate only a single spike at the onset of depolarization. This single spiking is in contrast with the repetitive firing of the M cells morphologically homologous RSNs, MiD2cm and MiD3cm, which are also involved in escapes. However, how the unique firing property of M cells is established and the underlying molecular mechanisms remain unclear. In the present study, we first demonstrated that the single-spiking property of M cells was acquired at 4 days postfertilization (dpf), accompanied by an increase in dendrotoxin I (DTX)-sensitive low-threshold K(+) currents, prior to which the M cell repetitively fires as its homologs. Second, in situ hybridization showed that among DTX-sensitive Kv1 channel α-subunits, zKv1.1a was unexpectedly expressed even in the homologs and the bursting M cells at 2 dpf. In contrast, zKvβ2b, an auxiliary β-subunit of Kv1 channels, was expressed only in the single-spiking M cells. Third, zKv1.1a expressed in Xenopus oocytes functioned as a low-threshold K(+) channel, and its currents were enhanced by coexpression of zKvβ2b subunits. Finally, knockdown of zKvβ2b expression in zebrafish larvae resulted in repetitive firing of M cells at 4 dpf. Taken together, these results suggest that associative expression of Kvβ2 subunits with Kv1.1 channels is crucial for developmental acquisition of the unique firing properties of the M cells among homologous neurons.

Comprehensive detection of viruses in pediatric patients with acute liver failure using next-generation sequencing

BACKGROUND Pediatric acute liver failure (PALF) is a rare and severe syndrome that frequently requires liver transplantation. Viruses are one of the most frequent causes of this disease, however, pathogenic viruses are not determined in many patients. Recently next-generation sequencing (NGS) has been applied to comprehensively detect pathogens of infectious diseases of unknown etiology. OBJECTIVES To evaluate an NGS-based approach for detecting pathogenic viruses in patients with PALF or acute hepatitis of unknown etiology. STUDY DESIGN To detect virus-derived DNA and RNA sequences existing in sera/plasma from patients, both DNA and RNA sequencing were performed. First, we validated the ability of NGS to detect viral pathogens in clinical serum/plasma samples, and compared different commercial RNA library preparation methods Then, serum/plasma of fourteen patients with PALF or acute hepatitis of unknown etiology were evaluated using NGS. RESULTS Among three RNA library preparation methods, Ovation RNA-Seq System V2 had the highest sensitivity to detect RNA viral sequences. Among fourteen patients, sequence reads of torque teno virus, adeno-associated virus, and stealth virus were found in the sera of one patient each, however, the pathophysiological role of these three viruses was not clarified. Significant virus reads were not detected in the remaining 11 patients. CONCLUSIONS This finding might be due to low virus titer in blood at the time of referral or a non-infectious cause might be more frequent. These results suggest an NGS-based approach has potential to detect viral pathogens in clinical samples and would contribute to clarification of the etiology of PALF.

Identification of potential pathogenic viruses in patients with acute myocarditis using next-generation sequencing: TAKEUCHI et al.

Myocarditis is an inflammatory disease of the myocardium and leads to cardiac dysfunction and heart failure. Although viral infections are considered to be the most common etiology of myocarditis, the identification of the causative virus is still challenging. Recently, next‐generation sequencing (NGS) has been applied in the diagnosis of infectious diseases. The aim of the current study was to comprehensively analyze potential pathogenic microorganisms using NGS in the sera of patients with myocarditis. Twelve pediatric and five adult patients hospitalized for acute myocarditis were included. Serum samples in the acute phase were obtained and analyzed using NGS to detect pathogen‐derived DNA and RNA. Viral sequence reads were detected in 7 (41%) of the 17 myocarditis patients by NGS. Among these patients, detection of Epstein‐Barr virus, human parvovirus B19, torque teno virus, and respiratory syncytial virus reads by NGS was consistent with polymerase chain reaction or antigen test results in one patient each. A large number of human pegivirus reads were detected from one patient by RNA sequencing; however, its pathogenicity to human is unknown. Conversely, the number of detected virus‐derived reads was small in most cases, and the pathophysiological role of these viruses remains to be clarified. No significant bacterial or fungal reads other than normal bacterial flora was detected. These data indicate that comprehensive detection of virus‐derived DNA and RNA using NGS can be useful for the identification of potential pathogenic viruses in myocarditis.

Comprehensive detection of pathogens in immunocompromised children with bloodstream infections by next-generation sequencing

Abstract Background Bloodstream infection (BSI) is a severe complication in immunocompromised patients. Prompt identification of causative microorganisms would improve the outcome of BSI due to optimization of antimicrobial treatment. Next-generation sequencing (NGS) allows us to analyze comprehensively and quantitatively all microorganisms present in a clinical sample in comparison with blood culture. However, there are currently no established methods to identify causative pathogens by NGS. Methods BSI was defined by the following criteria: (i) pathogen isolated from blood culture and (ii) fever ≥38.0°C or C-reactive protein >1.0mg/dl. Thirty-five pediatric patients (12 with BSI and 23 with suspected BSI/negative blood culture) were enrolled. Plasma/serum samples were used for sequencing and the results were compared with those from blood culture. The bacterial reads per million reads of the sequence depth (BR) and relative importance values of the dominant bacteria (P1) were applied to identify causative pathogens. Results Sequencing reads of bacteria isolated in blood culture were identified by NGS in all plasma/serum samples at the onset of BSI. Additionally, bacteria isolated in blood culture were identical to the dominant bacteria by NGS in 8 of 12 patients with BSI. Causative microorganisms were detected when the NGS results fulfilled the criteria of BR >200 and P1 >0.5. In two patients with catheter-related BSI, causative bacteria were detected in the plasma/serum at 7 days before disease onset. Causative pathogens (Tatlockia micdadei, Escherichia coli, and human adenovirus 2) were identified in three of 23 patients in the suspected BSI group. A total of 62 resistance genes were detected in nine patients with sequences covering 5–100% of references. Conclusion An NGS-based approach has great potential for analysis of causative microorganisms in BSI and may help to diagnose a disease before disease onset. Antimicrobial resistance genes can also be found through sequence data processing. Disclosures All authors: No reported disclosures.

Molecular basis for developmental acquisition of unique firing property of Mauthner cell in zebrafish

O3-J-2-1 Mechanistic basis of the bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium Takayuki Michikawa 1,2,3 , Tadashi Shinohara 2, Masahiro Enomoto 2, Jun-Ichi Goto 2, Miwako Iwai 4, Toru Matsu-ura 2, Haruka Yamazaki 2, Akitoshi Miyamoto 2, Akio Suzuki 2, Katsuhiko Mikoshiba 2,3 1 Lab. Mol. Neurogenesis, RIKEN Brain Sci. Inst., Wako, Japan 2 Lab. Dev. Neurobiol., RIKEN Brain Sci. Inst., Wako, Japan 3 Calcium Oscillation Project, ICORP-SORST, JST, Kawaguchi, Japan 4 Div. Mol. Phathol., Inst. Med. Sci., Univ. Tokyo, Tokyo, Japan

Inputs to areas around the superior temporal cortex of marmosets

Areas in and around superior temporal sulcus (STS) are thought to be important to social behavior (e.g., recognizing other’s action and intention). Although new-world monkey marmosets have strong pro-social nature, data related connection of STS is scanty. We performed retrograde tracer (CTB-Alexa 488 or 555) injection around superior temporal cortex of the marmosets. So far, we have made three injections into dorsal and ventral parts of middle STS and dorsal part of caudal STS. We found each injection resulted in unique input pattern, like macaque monkey (Selzer and Pandya, 1994). But, in general, areas around STS receive strong projection form multiple sources (insular, parietal cortex, frontal and occipital cortices), as expected as its multi-modal nature. Based on these results, we will discuss the subdivision of the marmoset areas around STS, by combining with electrophysiology and cytoarchitectures. Research fund: KAKENHI (22300104).