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Abstract This was the first longitudinal study to analyze dental clinic wastewater to estimate asymptomatic SARS-CoV-2 infection trends in children. We monitored wastewater over a 14-month period, spanning three major COVID-19 waves driven by the Alpha, Delta, and Omicron variants. Each Saturday, wastewater was sampled at the Pediatric Dental Clinic of the only dental hospital in Japan’s Saitama Prefecture. The relationship between the weekly number of cases in Saitama Prefecture among residents aged

Background: Acute gastroenteritis is one of the major causes of morbidity and mortality in young children worldwide. Among these, rotavirus, norovirus, and adenovirus have been reported as the primary viral pathogens associated with the disease. Rapid diagnosis of viral pathogens is crucial when diarrhea outbreaks occur to ensure the timely administration of appropriate treatment and control measures. Methods: We evaluated three immunochromatographic test kits designed for the detection of norovirus, rotavirus, and adenovirus in 71 stool specimens collected from children with diarrhea who visited clinics in Japan. The first kit is a triplex immunochromatographic test kit designed for simultaneous detections of norovirus, rotavirus, and adenovirus on a single strip (this kit was referred to as IC-A). The other two immunochromatographic test kits are a dual detection kit for rotavirus and adenovirus, and a single detection kit for norovirus (IC-B). The RT-PCR/PCR was used as the gold standard method. Results: The results revealed that both IC-A and IC-B kits exhibited the same level of sensitivity of detection for rotavirus (72.7%) and adenovirus (22.7%), although the detection rate was lower than that of the RT-PCR/PCR method. However, there was a slight difference in the sensitivity of detection for norovirus between IC-A and IC-B, at 86.7% and 93.3%, respectively. The sensitivity of detection for adenovirus of both kits was relatively lower than those of RT-PCR method. This could be due to low viral load of adenovirus in clinical specimens below the detection limit of IC-A and IC-B kits. However, both immunochromatographic test kits (IC-A and IC-B) exhibited 100% specificity for norovirus, rotavirus, and adenovirus. Conclusions: The triplex immunochromatographic test kit (IC-A) designed for simultaneous detection of norovirus, rotavirus, and adenovirus has been proved to be more practical and convenient than the use of single or dual detection kits with more or less the same sensitivity and specificity of detections.

Background: Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial for ending the pandemic of coronavirus disease 2019 (COVID-19). Currently, the cumulative effect of booster shots of mRNA vaccines on adverse events is not sufficiently characterized. Methods: A survey-based study on vaccine adverse events was conducted in a Japanese medical institute after the third dose of Pfizer BNT162b2. Adverse events were grouped using network analysis, and a heteroscedastic probit model was built to analyse adverse events. Results: There were two main clusters of adverse events, systemic and local injection site-associated events. Subject background and the experience of previous vaccine-related adverse events were variably associated with the occurrence and intensity of adverse events following the third dose. Among adverse events, only lymphadenopathy increased prominently following the third dose, while the largest increase in other systemic adverse events occurred generally following the second dose. Conclusions: The effect of repeated booster vaccines on the frequency and intensity of adverse events differs depending on the kind of adverse event.

Background: Human astrovirus (HAstV) infection is one of the leading causes of acute gastroenteritis in young children. The present study reports the outbreak of HAstV in children with acute gastroenteritis in Kyoto, Japan, during the COVID-19 pandemic, 2021. Methods: A total of 61 stool samples were collected from children with acute gastroenteritis who visited a pediatric outpatient clinic in Maizuru city, Kyoto, Japan from July to October, 2021. HAstV was screened by RT-PCR, and the genotypes were identified by nucleotide sequence analysis. Results: Of 61 cases of acute gastroenteritis, 20 were mono-infected with HAstV alone. In addition, mixed infection of HAstV and NoV, and HAstV and RVA were also detected in 15 and 1 cases, respectively. Of 36 HAstV strains detected in this outbreak, 29 and 7 were HAstV1 and MLB2 genotypes, respectively. All HAstV1 strains were closely related to the HAstV1 reported from Thailand and Japan in 2021 and all of them belonged to subgenotype HAstV1a. Among MLB2, they were most closely related to the MLB2 strains reported from China in 2016 and 2018. Conclusions: After the kindergartens and schools were re-opened at the middle of 2021 in Japan, an outbreak of HAstV was reported. Control measures against the COVID-19 pandemics might affect the spread of diarrheal virus infection. Here we report the outbreak of HAstV1 and MLB2 in Kyoto, Japan, during COVID-19 pandemic in 2021.

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013–2016) remained phylogenetically distinct from later strains (2019–2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment.

ABSTRACT Human astroviruses (HAstVs) are important causative pathogens of acute gastroenteritis (AGE) in children worldwide. MLB and VA HAstVs, which are genetically distinct from the previously known classic HAstVs, have been detected since 2008. To investigate the role of HAstVs in AGE, we conducted molecular detection and characterization of HAstVs circulating in children with AGE in Japan from 2014 to 2021. Out of 2,841 stool samples, HAstVs were detected in 130 (4.6%). MLB1 was the predominant genotype detected (45.4%), followed by HAstV1 (39.2%), MLB2 (7.4%), VA2 (3.1%), HAstV3 (2.3%), HAstV4, HAstV5, and MLB3 (0.8% each). The results demonstrated that HAstV infection in pediatric patients in Japan was dominated by the two major genotypes MLB1 and HAstV1, with a small proportion of other genotypes. The overall infection rates of MLB and VA HAstVs were higher than those of classic HAstVs. The HAstV1 strains detected in this study belonged solely to lineage 1a. The rare MLB3 genotype was detected for the first time in Japan. All three HAstV3 strains belonged to lineage 3c based on the ORF2 nucleotide sequence and were shown to be recombinant strains. IMPORTANCE HAstVs are one of the pathogens of viral AGE and are considered the third most common viral agents of AGE after rotavirus and norovirus. HAstVs are also suspected to be the causative agents of encephalitis or meningitis in immunocompromised patients and elderly persons. However, little is known about the epidemiology of HAstVs in Japan, especially that of MLBs and VA HAstVs. This study demonstrated epidemiological features and molecular characterization of human astroviruses encompassing a 7-year study period in Japan. This study highlights the genetic diversity of HAstV circulating in pediatric patients with acute AGE in Japan.

Background: Rotavirus A (RVA) is a major cause of severe acute gastroenteritis (AGE) in infants and children worldwide. In Japan, two kinds of rotavirus vaccines have been introduced as voluntary vaccines in 2011 and 2012, respectively, and launched into the national vaccine program in October 2020. Methods: In this study, we investigated prevalence of RVA and their molecular characterization in the stool samples collected from infants and children with AGE who visited one outpatient clinic in Japan, from July 2014 to June 2020, during voluntary vaccination with two kinds of rotavirus vaccines. Results: The RVA detection rates decreased from 44.7 % in 2014–2015 to 35.4 % in 2018–2019, whereas in 2019–2020 the numbers of samples collected were dramatically decreased and none of RVA was detected. During this study period, rotavirus vaccination rates in this area increased from 32.4 % to 62.2 %. Distribution of RVA VP7 (G), VP4 (P), and VP6 (I) genotypes in this area had changed year by year; the major genotype combinations were G1P[8]I1 and G1P[8]I2 in 2014–2015, G2P[4]I2 and G9P[8]I1 in 2015-2016, G1P[8]I1 and G8P[8]I2 in 2017–2018, and G8P[8]I2 in 2018–2019. Phylogenetic analysis demonstrated that VP7 nucleotide sequences of G1 were genetically diverse compared with those of other G genotypes in this study. Meanwhile, predominance of unusual G2P[8]I1, G2P[8]I2 and mixed P genotypes were observed only in 2016–2017, but did not carry on in 2017–2019. The equine-like G3 was detected only in 2016–2017. Conclusions: The results revealed diversity of RVA genotypes and the genotype combinations have changed year by year in Japan, during the study period of 2016–2020.

Background: Human sapovirus (SaV) is an important etiologic agent of childhood diarrhea. This study aims to investigate the burden of SaV infection in childhood diarrhea in Japan from 2009–2019, to understand the changes in SaV infection after the introduction of rotavirus (RV) vaccination in Japan in 2011. Methods: Stool samples were collected from children aged ≤ 12 years old with acute gastroenteritis (AGE) who visited outpatient clinics of six prefectures in Japan. The viral RNA was detected by RT-PCR and genogroups and genotypes were determined through sequence-based analysis. Results: Among 5697 stool samples, 318 (5.6%) samples remained SaV-positives showing the highest prevalence in June and 12–24 month aged children. The most predominant genotype was GI.1 (56.8%), followed by GI.2 (19.2%), GII.1 (10.8%), GIV.1 (9.4%), GI.3 (1.7%), GII.2 (1.4%), GII.3 and GII.5 (0.3%). Importantly, an increasing trend (P = 0.016) of SaV infection was observed during this period. In particular, SaV-detection rate was increased significantly (P = 0.033) from 4.3% in pre-rotavirus (RV)-vaccination era to 6.1% in post-RV-vaccination era. We provided evidence that this increase in SaV infection was mainly attributed by coinfections. Conclusions: The upward trend of SaV infection, particularly after the introduction of RV-vaccination, is an emerging concern. Attention should be paid to control this upward trend of SaV infection to ensure maximum benefits of implementation of RV vaccines towards reducing overall childhood diarrhea worldwide.

The female reproductive tract (FRT) and remote/versatile organs in the body share bidirectional communication. In this review, we discuss the framework of the “FRT-organ axes.” Each axis, namely, the vagina-gut axis, uterus-gut axis, ovary-gut axis, vagina-bladder axis, vagina-oral axis, uterus-oral axis, vagina-brain axis, uterus-brain axis, and vagina-joint axis, is comprehensively discussed separately. Each axis could be involved in the pathogenesis of not only gynecological diseases but also diseases occurring apart from the FRT. Although the microbiota is clearly a key player in the FRT-organ axes, more quantitative insight into the homeostasis of the microbiota could be provided by host function measurements rather than current microbe-centric approaches. Therefore, investigation of the FRT-organ axes would provide us with a multicentric approach, including immune, neural, endocrine, and metabolic aspects, for understanding the homeostatic mechanism of women’s bodies. The framework of the FRT-organ axes could also provide insights into finding new therapeutic approaches to maintain women’s health.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (blaKPC, blaNDM-1, blaIMP-1 group, and blaVIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

Although two live oral rotavirus (RV) vaccines, Rotarix and RotaTeq, play a critical role toward reducing disease severity, hospitalization, and death rate in RV infections, regular monitoring of vaccine effectiveness (VE) is yet necessary because the segmented genome structure and reassortment capability of RVs pose considerable threats toward waning VE. In this study, we examined the VE by a test-negative study design against G9P[8]I2 strain during a seasonal outbreak in February–May, 2018, in an outpatient clinic in Kyoto Prefecture, Japan. It remains important because G9P[8]I2 strain remains partially heterotypic to these vaccines and predominating in post-vaccination era. During year-long surveillance, RV infections were detected only from February to May. During this outbreak, 33 (42.3%) children out of 78 with acute gastroenteritis (AGE) remained RV-positive, of which 29 (87.8%) children were infected with G9P[8]I2. Two immunochromatographic (IC) assay kits exhibited 100% sensitivity and specificity to detect G9P[8]I2 strain. Only 23.2% children were found to be vaccinated. Yet, significant VE 69.7% (95% CI: 2.5%-90.6%) was recognized against all RV strains that increased with disease severity. Similar significant VE 71.8% (95% CI: 1%-92%) was determined against G9P[8]I2 strain. The severity score remained substantially low in vaccinated children. Our data reveal that vaccine-preventable G9P[8]I2 strain yet may cause outbreak where vaccination coverage remains low. Thus, this study emphasizes the necessity of global introduction of RV-vaccines in national immunization programs of every country.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100–200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500–3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Abstract Congenital metabolic diseases are a group of hereditary disorders caused by the deficiency of a single specific enzyme activity. Without appropriate therapy, affected patients suffer severe neurologic disability and eventual death. The current mainstays of management attempt to slow disease progression, but are not curative. Several of these diseases have demonstrated significant benefits from liver transplantation; however, this approach is limited by the morbidity associated with this invasive procedure and a shortage of donor organs. Therefore, there is a need to establish a new strategy for improving the quality of a life for these patients. One potential solution is regenerative therapy using hepatocytes generated from stem cells. Herein, we discuss pertinent issues necessary for clinical application of the human amniotic epithelial cell, a type of placental stem cell. Focusing on maple syrup urine disease as an example, where liver replacement is an effective therapy, we explore this approach from a clinician's perspective.

Rubella virus (RuV) infections in pregnant women, especially first-trimester infections, can lead to congenital rubella syndrome (CRS). However, the mechanisms of fetal RuV infection are not completely understood, and it is not observed in every pregnant woman infected with RuV. As gestational diabetes mellitus is a risk factor for congenital viral infections, we investigated the possible roles of hypoglycemia-related endoplasmic reticulum (ER) stress as a key factor for vertical RuV infection using immortalized human first-trimester trophoblasts. Low-glucose stress was induced prior to RuV infection by culturing HTR-8/SVneo and Swan.71 cells in low-glucose (LG) medium for 24 h or high-glucose medium for 6 h and then LG medium for an additional 18 h. Clinically isolated RuV was inoculated at a multiplicity of infection of 5 to 10. The intracellular localization of the RuV capsid protein was investigated 24 to 48 h post-infection (pi) with flow cytometry (FCM) analysis and fluorescence microscopy. Viral progeny production was monitored by FCM analysis. Increases in RuV infection in LG-induced ER-stressed trophoblasts were observed. No significant increase in apoptosis of RuV-infected cells was noted at days 2 and 5 pi, and substantial viral progeny production was observed until day 5 pi. An approximate fivefold increase in viral binding was noted for the LG-stressed cells. Although the detailed mechanisms underlying viral entry into LG-stressed cells are not known and require further investigation, these findings suggest that a certain degree of LG stress in early pregnancy may facilitate infection and cause CRS.

Many different enteric viruses can cause acute gastroenteritis in humans worldwide. While a single virus can indeed cause disease, multiple-virus infections are commonly reported. However, data regarding a comparison between single- and multiple-virus infections upon clinical manifestations of acute gastroenteritis are relatively limited. In this study, a total of 2383 fecal specimens were collected from children with acute gastroenteritis during June 2014–July 2017 in a pediatric clinic in Japan and tested for 11 viruses by multiplex RT-PCR. At least 1 virus was found in 1706 (71.6%) specimens and norovirus GII was the most frequent agent, followed by rotavirus A and other viruses. Multiple-virus infections were identified in 565 cases (33.1%). While major clinical symptoms were found to be significantly different in some single- vs. multiple-virus infections, the disease severity was statistically non-significant. Our study highlights the burden of multiple-virus infections for acute gastroenteritis and the clinical features of patients with multiple-virus infections.

[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (

Pregnant women presumably gather information about the coronavirus disease 2019 (COVID-19) from various sources. However, it is difficult for pregnant women who are not medical professionals to source the appropriate information because of the infodemic related to the COVID-19 pandemic. Therefore, the objective of our study was to investigate how pregnant women gathered information about COVID-19 and COVID-19 vaccination. To address this issue, we conducted an online questionnaire survey between 5 October and 22 November 2021, which was approved by the Ethics Committee of Nihon University School of Medicine. We received 4962 responses after excluding 1179 insufficient answers. Our study found that age, occupation, and infection-risk anxiety influenced the selection of media for obtaining information. Pregnant women who were older, medical professionals, public servants, or educators tended to rely on specialized medical websites, whereas housewives tended to use mass media, social media, and sources with uncertain scientific evidence. Additionally, the number of weeks of gestation and the method of conception (natural or assisted reproductive conception) affected the selection of media. The accessibility of COVID-19 information for pregnant women was determined by their social background and pregnancy status. We need to continue making efforts to ensure that appropriate information is readily available to pregnant women and their families.

Bacteraemia brought on by Clostridium perfringens has a very low incidence but is severe and fatal in fifty per cent of cases. C. perfringens is a commensal anaerobic bacterium found in the environment and in the intestinal tracts of animals; it is known to produce six major toxins: α-toxin, β-toxin, ε-toxin, and others. C. perfringens is classified into seven types, A, B, C, D, E, F and G, according to its ability to produce α-toxin, enterotoxin, and necrotising enterotoxin. The bacterial isolates from humans include types A and F, which cause gas gangrene, hepatobiliary infection, and sepsis; massive intravascular haemolysis (MIH) occurs in 7–15% of C. perfringens bacteraemia cases, resulting in a rapid progression to death. We treated six patients with MIH at a single centre in Japan; however, unfortunately, they all passed away. From a clinical perspective, MIH patients tended to be younger and were more frequently male; however, there was no difference in the toxin type or genes of the bacterial isolates. In MIH cases, the level of θ-toxin in the culture supernatant of clinical isolates was proportional to the production of inflammatory cytokines in the peripheral blood, suggesting the occurrence of an intense cytokine storm. Severe and systemic haemolysis is considered an evolutionary maladaptation as it leads to the host’s death before the bacterium obtains the benefit of iron utilisation from erythrocytes. The disease’s extraordinarily quick progression and dismal prognosis necessitate a straightforward and expedient diagnosis and treatment. However, a reliable standard of diagnosis and treatment has yet to be put forward due to the lack of sufficient case analysis.

Transforming growth factor-beta 1 (TGF-β1) is a pleiotropic growth factor playing various roles in the human body including cell growth and development. More functions of TGF-β1 have been discovered, especially its roles in viral infection. TGF-β1 is abundant at the maternal–fetal interface during pregnancy and plays an important function in immune tolerance, an essential key factor for pregnancy success. It plays some critical roles in viral infection in pregnancy, such as its effects on the infection and replication of human cytomegalovirus in syncytiotrophoblasts. Interestingly, its role in the enhancement of Zika virus (ZIKV) infection and replication in first-trimester trophoblasts has recently been reported. The above up-to-date findings have opened one of the promising approaches to studying the mechanisms of viral infection during pregnancy with links to corresponding congenital syndromes. In this article, we review our current and recent advances in understanding the roles of TGF-β1 in viral infection. Our discussion focuses on viral infection during pregnancy, especially in the first trimester. We highlight the mutual roles of viral infection and TGF-β1 in specific contexts and possible functions of the Smad pathway in viral infection, with a special note on ZIKV infection. In addition, we discuss promising approaches to performing further studies on this topic.

Cow milk is a nourishing food containing numerous essential nutrients. In Japan, the consumption of cow milk is thought to enhance resistance to exhaustion-related diseases. Although several nutrients in cow milk, such as lactoferrin, are thought to modulate immune cells, the mechanisms remain unclear. Recently, the immunoregulatory functions of food-derived microRNAs or exosomes have been reported. Therefore, we studied the effects of exosomes derived from cow milk (CM-Exs) on immune cells in the present study. We obtained blood samples from healthy adult donors with the approval of the ethics committee. Peripheral blood mononuclear cells (PBMCs) were stimulated with CM-Exs in the absence or presence of interleukin-2 (IL-2) and IL-12. Cell surface markers and intracellular cytokine production were analysed by flow cytometry. CM-Ex stimulation enhanced the expression of CD69 on NK cells. Although CM-Ex stimulation alone did not induce interferon-γ (IFN-γ) production by NK cells or γδT cells, simultaneous stimulation with CM-Ex, IL-2 and IL-12 significantly enhanced IFN-γ production. In conclusion, cow milk consumption alone may not activate immune cells; however, CM-Exs could enhance immune cells under inflammatory conditions.

Background: Enteric caliciviruses, including noroviruses (NoVs) and sapoviruses (SaVs) are the most significant pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. Methods: In this study, 126 environmental water samples collected from 6 different sources in Chiang Mai, Thailand from November 2016 to July 2018 were examined for the presence of genogroups I, II, IV (GI, GII, GIV) NoVs and SaVs by using RT-nested PCR assays, genome sequencing, and phylogenetic analysis, Results: Forty out of 126 (31.7%) water samples were positive for one or more caliciviruses throughout the years of study with high prevalence in winter. Among 126 tested specimens, 34 (27.0%), 30 (23.8%), 3 (2.4%), and 2 (1.6%) were positive for NoV GI, GII, GIV, and SaV, respectively. For NoV GI, 6 different genotypes were identified with the most predominant of GI.1 genotype (17 strains). In addition, 6 different genotypes of GII were detected with high prevalence of GII.17 (12 strains) and GII.2 (11 strains). It was interesting to note that our study reported the detection of NoV GIV for the first time in water samples in Thailand, and all were GIV.1 genotype. For SaV detection, only 2 water samples were positive for SaV GI. Conclusions: The data revealed heterogeneity and highly dynamic distribution of NoV GI, GII, GIV, and SaV in environmental water in Chiang Mai, Thailand, during the study period of 2016–2018.

Abstract The threat of predation is a driving force in the evolution of animals. We have previously reported that Xenopus laevis enhanced their tail muscles and increased their swimming speeds in the presence of Japanese larval salamander predators. Herein, we investigated the induced gene expression changes in the brains of tadpoles under the threat of predation using 3′-tag digital gene expression profiling. We found that many muscle genes were expressed after 24 h of exposure to predation. Ingenuity pathway analysis further showed that after 24 h of a predation threat, various signal transduction genes were stimulated, such as those affecting the actin cytoskeleton and CREB pathways, and that these might increase microtubule dynamics, axonogenesis, cognition, and memory. To verify the increase in microtubule dynamics, DiI was inserted through the tadpole nostrils. Extension of the axons was clearly observed from the nostril to the diencephalon and was significantly increased (P ≤ 0.0001) after 24 h of exposure to predation, compared with that of the control. The dynamic changes in the signal transductions appeared to bring about new connections in the neural networks, as suggested by the microtubule dynamics. These connections may result in improved memory and cognition abilities, and subsequently increase survivability.

The roles of interleukin-22 (IL-22) in carcinogenesis have been proposed in various neoplasms. Increased expression of IL-22 has been observed in oral squamous cell carcinoma (OSCC) lesions as well as in other cancers. OSCC is still associated with poor prognosis and a high mortality rate because of its invasiveness and frequent lymph node metastasis. In the present study, we investigated the effects of IL-22 on OSCC cells. The human OSCC cell lines Ca9-22 and SAS were stimulated with IL-22 (1–10 ng/mL), and their migration abilities were examined using a cell scratch assay. A Matrigel invasion assay was performed to evaluate the invasion abilities of OSCC cells. Signal transducer and activator of transcription 3 (STAT3) phosphorylation, matrix metalloproteinase (MMP) and epithelial-mesenchymal transition (EMT)-related genes and proteins were also examined. IL-22 treatment promoted the migration and invasion abilities of OSCC cells without increasing their viability. IL-22 stimulation also induced STAT3 phosphorylation, MMP-9 activity and EMT-related genes and proteins. Our findings suggest that IL-22 has possible roles in the development of OSCC.

The surfaces of commercially available pure titanium (cpTi) and Ti6Al4V alloy specimens were modified to form highly ordered rutile nanorod arrays by chemical treatment and subsequent aging treatment. The densities of the rutile rods were (1.04 ± 0.06) ×103 and (0.70 ± 0.10) ×103 μm–2 for the cpTi and Ti6Al4V alloy specimens, respectively. Both the rutile nanorod arrays on the cpTi and Ti6Al4V alloy specimens deposited apatite particles when soaked in simulated body fluid (SBF) for one day. After soaking for various other periods, scanning electron microscopy images and thin-film X-ray diffraction patterns of these specimens showed that the cpTi specimens exhibited a superior rate of apatite nucleation and favored the formation of numerous apatite particles with larger diameter. This superior apatite-forming ability of the cpTi specimens can be attributed to the dense, thick titania layers with higher rutile nanorod density on their surfaces.

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Mitochondrial diabetes (MD) is generally classified as a genetic defect of β-cells. The main pathophysiology is insulin secretion failure in pancreatic β-cells due to impaired mitochondrial ATP production. However, several reports have mentioned the presence of insulin resistance (IR) as a clinical feature of MD. As mitochondrial dysfunction is one of the important factors causing IR, we need to focus on IR as another pathophysiology of MD. In this special issue, we first briefly summarized the insulin signaling and molecular mechanisms of IR. Second, we overviewed currently confirmed pathogenic mitochondrial DNA (mtDNA) mutations from the MITOMAP database. The variants causing diabetes were mostly point mutations in the transfer RNA (tRNA) of the mitochondrial genome. Third, we focused on these variants leading to the recently described “tRNA modopathies” and reviewed the clinical features of patients with diabetes. Finally, we discussed the pathophysiology of MD caused by mtDNA mutations and explored the possible mechanism underlying the development of IR. This review should be beneficial to all clinicians involved in diagnostics and therapeutics related to diabetes and mitochondrial diseases.

Mycobacterium tuberculosis (Mtb) infection remains a major health problem worldwide. Although the Bacillus Calmette-Guérin (BCG) vaccine is the most widely used vaccination for preventing tuberculosis (TB), its efficacy is limited. We previously developed a new recombinant BCG (rBCG)-based vaccine encoding the Ag85B protein of M. kansasii (Mkan85B), termed rBCG-Mkan85B, and its administration is followed by boosting with plasmid DNA expressing the Ag85B gene (DNA-Mkan85B). Previously, we identified MHC-I (H2-Kd)-restricted epitopes that highly cross-react with those of Mtb in BALB/c (H2d) and CB6F1 (H2b/d) mice. We also reported that the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination protocol protected CB6F1 mice against M. kansasii infection. In this study, to investigate the protective effect of our novel rBCG against Mtb infection, CB6F1 mice were either left unimmunized or immunized with the BCG, rBCG-Mkan85B, or rBCG-Mkan85B/DNA-Mkan85B vaccine for 10 weeks prior to inhalation exposure to the virulent Mtb Erdman strain for another 6 weeks. Compared with the BCG and rBCG-Mkan85B vaccinations, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination protocol significantly reduced the numbers of pulmonary colony-forming units (CFUs). Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific polyfunctional CD4+ and CD8+ T cells. These results suggest that CD8+ T-cell immunity to immunodominant epitopes of Mtb is enhanced by rBCG vector-based immunization. Thus, rBCG vector-based vaccinations may overcome the limited ability of the current BCG vaccine to elicit TB immunity.

The Zika virus (ZIKV) is well known for causing congenital Zika syndrome if the infection occurs during pregnancy; however, the mechanism by which the virus infects and crosses the placenta barrier has not been completely understood. In pregnancy, TGF-β1 is abundant at the maternal–fetal interface. TGF-β1 has been reported to enhance rubella virus binding and infection in human lung epithelial cells. Therefore, in this study, we investigate the role of TGF-β1 in ZIKV infection in the immortalized human first-trimester trophoblasts, i.e., Swan.71. The cells were treated with TGF-β1 (10 ng/mL) for two days before being inoculated with the virus (American strain PRVABC59) at a multiplicity of infection of five. The results showed an enhancement of ZIKV infection, as demonstrated by the immunofluorescent assay and flow cytometry analysis. Such enhanced infection effects were abolished using SB431542 or SB525334, inhibitors of the TGF-β/Smad signaling pathway. An approximately 2-fold increase in the virus binding to the studied trophoblasts was found. In the presence of the Smad inhibitors, virus replication was significantly suppressed. An enhancement in Tyro3 and AXL (receptors for ZIKV) expression induced by TGF-β1 was also noted. The results suggest that TGF-β1 promotes the virus infection via the Smad pathway. Further studies should be carried out to clarify the underlying mechanisms of these findings.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.

Although autoclaving is a common sterilization method for biomedical devices, the ability to induce deposition of apatite particles on hydrothermally treated titanium is still not fully realized. This is because the induction ability is too weak to be evaluated via in vitro apatite formation in Kokubo’s simulated body fluid (SBF) by the conventional immersion method, i.e. using samples with open and smooth surface. This study reports on the surface structure of hydrothermally treated titanium and the ability to induce deposition of apatite particles on the surface of parallel confined spaces separated by sub-millimeter gaps in Kokubo’s SBF. Thin-film X-ray diffraction and analyses using Fourier transform infra-red (FT-IR) spectroscopy and Raman spectroscopy revealed that a nano-crystalline anatase-type titanium oxide layer was formed on titanium substrates after hydrothermal treatment at 150°C for 2 h. When growth of the titanium oxide layer was moderately suppressed, the hydrothermally treated titanium surface exhibited a characteristic interference color, silver or gold, which does not impair the esthetic appearance of the titanium-based implant. The ability to induce deposition of apatite particles on hydrothermally treated titanium was remarkably amplified by parallel alignment of substrates separated by sub-millimeter gaps.

Reports of invasive disease due to Streptococcus pneumoniae have declined since the introduction of pneumococcal conjugate vaccines (PCV7 and PCV13). The incidence of invasive diseases due to S. pneumoniae that are not addressed by the vaccines, however, has increased in children and adults, creating a global public health problem. Previously, we established the loop-mediated isothermal amplification (LAMP) method for a PCV13 serotype-specific assay. In the current study, we developed a rapid, simple, and cost-effective assay to detect serotypes in the 23-valent pneumococcal polysaccharide vaccine (PPSV23) using the LAMP method. In this study, LAMP primer sets for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F of S. pneumoniae were developed. The reactivity, specificity, and sensitivity of LAMP assays were determined and compared to those of conventional PCR. The feasibility of LAMP assays in clinical application in patients with invasive pneumococcal diseases was validated by defining the detection limit of the LAMP assay with bacterial genomic DNA-spiked blood specimens. The specificity of each LAMP assay was determined using 44 serotypes of pneumococcal strains. Their sensitivity was 100 copies per reaction versus 103 to 106 copies per reaction for PCR assays. Using DNA-spiked blood specimens, excluding the LAMP assay that targeted serotype 22F (103 copies per reaction), the limit of detection of the LAMP assay was similar to that with purified DNA as the template (102 copies per reaction), compared with 103 to >106 copies per reaction for PCR assays. In conclusion, a rapid and simple LAMP-based PPSV23-targeted serotype detection assay was developed for use in many countries. This study is the first report of a LAMP-based assay for identification of PPSV23 serotypes. Further evaluation of this assay is needed through surveillance and vaccine efficacy studies.

We recently published an article about myelin oligodendrocyte glycoprotein-independent rubella infection of keratinocytes in vitro, in which first-trimester trophoblast cells were shown as rubella virus (RuV)-resistant. Given an incident rate as high as 90% of congenital rubella syndrome in the first eight weeks of pregnancy, the RuV infection of first-trimester trophoblasts is considered key to opening the gate to transplacental transmission mechanisms. Therefore, with this study, we aimed to verify the susceptibility/resistance of first-trimester trophoblast cell lines, HTR-8/SVneo and Swan.71, against RuV. Cells cultured on multi-well plates were challenged with a RuV clinical strain at a multiplicity of infection from 5 to 10 for 3 h. The infectivity was investigated by immunofluorescence (IF) assay and flow cytometry (FCM) analysis. Supernatants collected during the post-infection period were used to determine virus-progeny production. The scattered signaling of RuV infection of these cells was noted by IF assay, and the FCM analysis showed an average of 4–5% of gated cells infected with RuV. In addition, a small but significant production of virus progeny was also observed. In conclusion, by employing appropriate approaches, we determined the low infectivity of RuV in first-trimester trophoblast cell lines but not resistance as in our previous report.

Chitosan is a cationic polymer that forms polymerized membranes upon reaction with anionic polymers. Chitosan−carboxymethyl cellulose (CMC) capsules are drug delivery carrier candidates whose mechanical strength and permeability must be controlled to achieve sustained release. In this study, the capsules were prepared from chitosan−γ-glycidoxypropyltrimethoxysilane (GPTMS)−CMC. The mechanical stability of the capsules was improved by crosslinking the chitosan with GPTMS. The capsules were then coated with hydroxyapatite (HAp) by alternately soaking them in calcium chloride solution and disodium hydrogen phosphate solution to prevent rapid initial drug release. Cytochrome C (CC), as a model drug, was introduced into the capsules via two routes, impregnation and injection, and then the CC released from the capsules was examined. HAp was found to be deposited on the internal and external surfaces of the capsules. The amount of CC introduced, and the release rate were reduced by the HAp coating. The injection method was found to result in the greatest CC loading.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner “What's in My Pot?” (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

The incidence of infections with nontuberculous mycobacteria (NTM) has been increasing worldwide. The emergence of multidrug-resistant NTM is a serious clinical concern, and a vaccine for NTM has not yet been developed. We previously developed a new recombinant Bacillus Calmette–Guérin (rBCG) vaccine encoding the antigen 85B (Ag85B) protein of Mycobacterium kansasii—termed rBCG-Mkan85B—which was used together with a booster immunization with plasmid DNA expressing the same M. kansasii Ag85B gene (DNA-Mkan85B). We reported that rBCG-Mkan85B/DNA-Mkan85B prime–boost immunization elicited various NTM strain-specific CD4+ and CD8+ T cells and induced Mycobacterium tuberculosis-specific immunity. In this study, to investigate the protective effect against M. kansasii infection, we challenged mice vaccinated with a rBCG-Mkan85B or rBCG-Mkan85B/DNA-Mkan85B prime–boost strategy with virulent M. kansasii. Although BCG and rBCG-Mkan85B immunization each suppressed the growth of M. kansasii in the mouse lungs, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination reduced the bacterial burden more significantly. Moreover, the rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination induced antigen-specific CD4+ and CD8+ T cells. Our data suggest that rBCG-Mkan85B/DNA-Mkan85B prime–boost vaccination effectively enhances antigen-specific T cells. Our novel rBCG could be a potential alternative to clinical BCG for preventing various NTM infections.

Abstract Background Delayed wound healing reduces the quality of life (QOL) of patients. Thus, understanding the mechanism of wound healing is indispensable for better management. However, the role of innate immunity in wound healing is thus far unknown. Recently the involvement of TLR3 in wound healing has been evaluated. The systemic administration of polyriboinosinic-polyribocytidylic acid (poly I:C ; a substitute for viral dsRNA and a ligand of toll-like receptor 3), enhances wound healing in vivo. The aim of this study is to improve our understanding of the link between innate immunity and human wound healing, particularly in re-epithelialization. Results The present study showed that poly I:C significantly accelerated collective HaCaT cell migration in a scratch assay. Poly I:C also increased IL-8 and bFGF production, and anti-IL-8 antibodies significantly inhibited the migration caused by poly I:C. Human recombinant IL-8 also accelerated collective HaCaT cell migration. An immunofluorescence assay and enzyme-linked immunosorbent assay (ELISA) also revealed that poly I:C decreased E-cadherin protein levels and increased vimentin protein levels, and anti-IL-8 antibody reversed this effect. In contrast, nucleic/cytosolic protein ratios of Snail 1 were unchanged in all tested conditions. Conclusion Our findings demonstrated that poly I:C accelerated collective HaCaT cell migration via autocrine/paracrine secretions of IL-8 and the subsequent incomplete epithelial-mesenchymal transition (EMT). Our findings provide a new strategy for wound healing by regulating innate immune systems in re-epithelialization.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (blaGES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of blaGES and another LAMP method to discriminate carbapenemase genotypes of blaGES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting blaGES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting blaGES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected blaGES with high sensitivity in all DNA-spiked samples; PCR did not detect blaGES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing blaGES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two blaGES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect blaGES and critical unique mutations.

Virus–host cell interactions in rubella virus (RuV) are of great interest in current research in the field, as their mechanism is not yet well understood. By hypothesizing that the epithelial-to-mesenchymal transition (EMT) may play a role in RuV infection, this study aimed to investigate the influence of TGF-β1-induced EMT of human lung epithelial A549 cells on the infectivity of RuV. A549 cells were cultured and treated with TGF-β1 for 1 to 2 days prior to virus infection (with a clinical strain). Viral infectivity was determined by flow cytometry analysis of cells harvested at 24 and 48 h post-infection (hpi) and by titration of supernatants collected at 48 hpi. The results showed that the percentages of the TGF-β1-treated A549 cells that were positive for RuV were at least twofold higher than those of the control, and the viral progeny titers in the supernatants collected at 48 hpi were significantly higher in the treatment group than in the control group. In addition, the virus binding assay showed a strong increase (more than threefold) in the percentages of RuV-positive cells, as determined by flow cytometry analysis and further confirmed by real-time PCR. Such an enhancement effect on RuV infectivity was abolished using LY364947 or SB431542, inhibitors of the TGF-β/Smad signaling pathway. The findings suggest that the TGF-β1-induced EMT-like process enhances RuV binding and infection in A549 cells via the Smad pathway. Further studies are necessary to identify possible proteins that facilitate viral binding and entry into treated cells.

Chitosan nanofiber has a highly uniform structure of 20–50 nm in diameter and shows high dispersibility in water due to its submicron size and high surface-to-volume ratio. The stacked nanofibers film is useful for breathability because it has a gap with a size of several tens of nm or more. However, the chemical bonds between the nanofibers cannot be broken during use. In this study, the thin films were obtained by filtration of chitosan nanofibers and 3-glycidoxypropyltrimethoxysilane (GPTMS) mixture. The addition of GPTMS changed the wettability, mechanical property and stability in water of the thin films. Bacitracin zinc salt (BZ) has been used for the localized dermatological medicines and loaded in the films. BZ interacted electrostatically with the thin films matrix and the release of BZ was controlled by the amount of GPTMS. A higher released amount of BZ showed higher antibacterial effects toward S. aureus. The film was also tested their toxicity by L929 fibroblasts. The release of less than 11.9 μg of BZ showed antibacterial effects, but were not toxic for fibroblast cells.

The rapid, accurate, and efficient identification of an infectious disease is critical to ensure timely clinical treatment and prevention in public health settings. In 2015, meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis was responsible for 379,200 (range: 322,700–444,700) deaths. Clinical features alone cannot determine whether bacterial meningitis is present; an analysis of cerebrospinal fluid (CSF) is essential. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method offering an alternative to polymerase chain reaction (PCR). LAMP-based assays for detection of three leading bacteria in CSF for diagnosis of meningitis have been established. The typing assays using LAMP for detection of meningococcal serogroups A, B, C, W, X, and Y as well as H. influenzae serotypes a, b, c, d, e, and f were launched. In comparative analysis of the meningitis pathogen assays, LAMP assays did not yield false negative results, and the detection rate of LAMP assays was superior compared with PCR or conventional culture methods. LAMP assays provide accurate and rapid test results to detect major bacterial meningitis pathogens. Accumulating evidence suggests that LAMP assays have the potential to provide urgently needed diagnostics for bacterial meningitis in resource-limited settings of both developed and developing countries.

Over the past four decades, the incidence of meningitis caused by Haemophilus influenzae in children has decreased due to widespread vaccination against H. influenzae type b (Hib). The incidence of invasive diseases due to H. influenzae types not included in the vaccines, however, has increased. At present, there are a limited number of diagnostics available to detect non-type b H. influenzae. To address this issue, we developed a rapid, simple, and cost-effective method for detecting serotypes of H. influenzae. We designed LAMP primer sets based on published sequences for H. influenzae capsular types a, c, d, e, and f. The assay was evaluated to determine test reactivity, specificity, and sensitivity. To support its use in patients with suspected meningitis, we evaluated the detection limit of the non-Hib serotype specific LAMP assay using bacterial genomic DNA-spiked cerebrospinal fluid (CSF) specimens. The reactivity and specificity of the LAMP assays were confirmed using six serotypes and non-typeable H. influenzae strains, plus eight strains of other Haemophilus species and non-Haemophilus genera. The detection limits of the LAMP assay for capsular types a, c, d, e, and f were 102, 102, 102, 103, and 10 copies per reaction, while those of the PCR assay were 104, 104, 103, 103, and 104 genome copies per reaction, respectively. Using DNA-spiked CSF specimens, the detection limit of the LAMP assay was equivalent to that using purified DNA as the template. However, the detection limit of the PCR was reduced from 103 to 104 genome copies per reaction for serotype d and from 103 to 105 genome copies per reaction for serotype e. To the best of our knowledge, this is the first report of a serotype-specific identification assay for H. influenzae using the LAMP method. Our results suggest the potential of LAMP methods for patients with suspected meningitis in resource-limited laboratories or public health surveillance systems.

Microporous spheres in a hybrid system consisting of chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) have advantages in a range of applications, e.g., as vehicles for cell transplantation and soft tissue defect filling materials, because of their excellent cytocompatibility with various cells. In this study, microporous chitosan-GPTMS spheres were prepared by dropping chitosan-GPTMS precursor sols, with or without a cerium chloride, into liquid nitrogen using a syringe pump. The droplets were then freeze dried to give the pores of size 10 to 50 μm. The cell culture tests showed that L929 fibroblast-like cells migrated into the micropores larger than 50 μm in diameter, whereas MG63 osteoblast-like cells proliferated well and covered the granule surfaces. The spheres with cerium chloride showed antibacterial properties against both gram-negative and gram-positive bacteria.

As part of the development of a flow-induced vibration evaluation methodology for the primary cooling piping in Japan sodium-cooled fast reactor, important factors were discussed in evaluating the flow-induced vibration for the hot-leg piping. To investigate a complex flow near the inlet of the hot-leg piping, a steady-state numerical analysis was carried out for the reactor upper plenum flow, which was simulated in a 1/10-scale reactor upper plenum experiment. Based on this analysis, experimental conditions on swirl inflow and deflected inflow that were identified as important factors were determined for flow-induced vibration experiments simulating only the hot-leg piping. In this study, the effect of the swirl inflow on flow pattern and pressure fluctuation onto the pipe wall was investigated in a 1/3-scale hot-leg pipe experiment. The experiment has indicated less significant for the pressure fluctuations, while the flow separation region was slightly influenced by the swirl inflow. Computational fluid dynamics simulation with a U-RANS approach results also appear in this paper. Through the simulations under the swirl inflow conditions of 0% and 5%, the validity of the U-RANS simulation was confirmed by comparison to the 1/3-scale hot-leg piping experiments.

Rubella virus (RuV), which belongs to the family Togaviridae and genus Rubivirus, causes systemic infection in children and young adults and congenital rubella syndrome in developing fetuses if the infection occurs during pregnancy. The mechanisms of fetal infection by RuV are not completely understood. Myelin oligodendrocyte glycoprotein (MOG) is reported to be a cellular receptor for RuV; however, it is mainly expressed in the central nervous system. Therefore, it is thought that other receptors are also responsible for virus entry into susceptible cells. In this study, we found that first-trimester trophoblast cells were resistant to RuV. In addition, we showed that HaCaT cells (an immortalized keratinocyte cell line) that did not express MOG on their surface were infected with RuV. This finding is one of the first demonstrations of MOG-independent RuV infection of susceptible host cells and suggests that it is important to continue searching for alternative RuV receptors. In addition, this study reports the resistance of first-trimester trophoblast cells to RuV and suggests that utilizing an epithelial–mesenchymal transition approach to study the mechanisms of transplacental vertical RuV infection.

Acute urinary bladder distension (AUBD) can activate bladder mechanical afferent and renal sympathetic nerves, which contributes to renal vasoconstriction. We hypothesized that AUBD-induced renal sympathetic activation may contribute to inflammatory responses and end-organ damage via activation of angiotensin-II-receptor-mediated intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte infiltration in the kidney. Methods: We evaluated the effect of 2 hours of AUBD induced by a threshold volume (micturition volume) on renal oxygen tension, microcirculation, renal reactive oxygen species (ROS) and monocyte/ macrophage (ED-1) infiltration, and ICAM-1 expression in the kidneys of urethane-anesthetized female Wistar rats. Bilateral ureteral dissection, renal denervation and intrarenal angiotensin II type 1 receptor blockade (2 mg/kg valsartan) were used to determine their roles in AUBD-induced renal oxidative stress. Results: Our results showed that AUBD evoked hypertension, a reduction in cortex oxygen tension and microcirculation, and increased renal ROS production, which were caused by increased perivascular and interstitial monocyte/macrophage infiltration and endothelial ICAM-1 overexpression. Renal denervation and angiotensin II type 1 receptor antagonist, but not bilateral ureter dissection, abolished the reduction in cortex oxygen tension and microcirculation, increased renal ROS production, increased perivascular monocyte/macrophage infiltration, and led to endothelial ICAM-1 overexpression in the kidney. Conclusion: Acute urinary retention enhances renal sympathetic activity, which causes renal vasoconstriction and increases oxidative stress, adhesion-molecule expression and leukocyte infiltration in the rat kidney via the angiotensin II type 1 receptor pathway.

Respiratory syncytial virus (RSV) is a major cause of acute respiratory infections (ARIs) in children worldwide and can cause high mortality, especially in developing countries. However, information on the clinical and molecular characteristics of RSV infection in developing countries is limited. From April 2010 to May 2011, 1,082 nasopharyngeal swabs were collected from children with ARI admitted to the Children's Hospital 2, Ho Chi Minh City, Vietnam. Samples were screened for RSV and genotyped by reverse transcription-PCR and sequencing. Demographic and clinical data was also recorded. RSV was found in 23.8% (257/1,082) of samples. RSV A was the dominant subgroup, accounting for 91.4% (235/257), followed by RSV B, 5.1% (13/257), and 9 cases (3.5%) were mixed infection of these subgroups. The phylogenetic analysis revealed that all group A strains belonged to the GA2 genotype. All group B strains belonged to the recently identified BA genotype, and further clustered into 2 recently described subgenotypes BA9 and BA10. One GA2 genotype strain had a premature stop codon which shortened the G protein length. RSV infection was significantly associated with younger age and higher severity score than those without. Co-infection with other viruses did not affect disease severity. RSV A caused more severe disease than RSV B. The results from this study will not only contribute to the growing database on the molecular diversity of RSV circulating worldwide but may be also useful in clinical management and vaccine development.

The third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism and host humoral immune responses. Positive charge of the V3 plays a key role in determining viral coreceptor tropism. Here, we examined by bioinformatics, experimental, and protein modelling approaches whether the net positive charge of V3 sequence regulates viral sensitivity to humoral immunity. We chose HIV-1 CRF01_AE strain as a model virus to address the question. Diversity analyses using CRF01_AE V3 sequences from 37 countries during 1984 and 2005 (n = 1361) revealed that reduction in the V3's net positive charge makes V3 less variable due to limited positive selection. Consistently, neutralization assay using CRF01_AE V3 recombinant viruses (n = 30) showed that the reduction in the V3's net positive charge rendered HIV-1 less sensitive to neutralization by the blood anti-V3 antibodies. The especially neutralization resistant V3 sequences were the particular subset of the CCR5-tropic V3 sequences with net positive charges of +2 to +4. Molecular dynamics simulation of the gp120 monomers showed that the V3's net positive charge regulates the V3 configuration. This and reported gp120 structural data predict a less-exposed V3 with a reduced net positive charge in the native gp120 trimer context. Taken together, these data suggest a key role of the V3's net positive charge in the immunological escape and coreceptor tropism evolution of HIV-1 CRF01_AE in vivo. The findings have molecular implications for the adaptive evolution and vaccine design of HIV-1.