Abstract
Hair follicle epithelial stem cells (HFSCs), which exist in the bulge region, have important functions for homeostasis of skin as well as hair follicle morphogenesis. Although several ...methods for isolation of HFSCs using a variety of stem cell markers have been reported, few investigations regarding culture methods or techniques to yield long-term maintenance of HFSCs in vitro have been conducted. In the present study, we screened different types of commercially available culture medium for culturing HFSCs. Among those tested, one type was shown capable of supporting the expression of stem cell markers in cultured HFSCs. However, both the differentiation potential and in vivo hair follicle-inducing ability of HFSCs serially passaged using that optimal medium were found to be impaired, probably because of altered responsiveness to Wnt signaling. The changes noted in HFSCs subjected to a long-term culture suggested that the Wnt signaling-related environment must be finely controlled for maintenance of the cells.
Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has ...yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.
Anisakiasis is one of the most common fishborne helminthic diseases in Japan, which is contracted by ingesting the larvae of the nematode Anisakis spp. carried by marine fish. Anisakis simplex sensu ...stricto (s.s.) and A. pegreffii are the dominant species in fish caught offshore Japan. The present study aimed to identify the anisakid species infecting Japanese patients and determine whether there is any difference in the pathogenetic potential of A. simplex (s.s.) and A. pegreffii. In total, 41 and 301 Anisakis larvae were isolated from Japanese patients and chub mackerel (Scomber japonicus), respectively; these were subjected to molecular identification using polymerase chain reaction targeted at a ribosomal DNA internal transcribed spacer region. Chub mackerel larvae were further examined for survival in artificial gastric juice (pH 1.8) for 7 days and for invasiveness on 0.75% solid agar over a 24-h interval. All clinical isolates, including those of asymptomatic, acute, and chronic infections as well as those from the stomach, small intestine, colon, and stool, were identified as A. simplex (s.s.). Chub mackerel harbored A. simplex (s.s.) and A. pegreffii larvae, together with a few larvae of other anisakid species. A. simplex (s.s.) larvae from chub mackerel tolerated the artificial gastric juice better than A. pegreffii, with 50% mortality in 2.6 and 1.4 days, respectively. In addition, A. simplex (s.s.) penetrated the agar at significantly higher rates than A. pegreffii. These results show that A. simplex (s.s.) larvae have the potential to survive acidic gastric juice to some extent and penetrate the stomach, small intestine, or colon in infected humans.
•S. mansoni larvae changes their location in snails after infection.•Miracidia infect through the head-foot (HF) area of snail.•Predominant migration to the hepatopancreas and ovotestis (HPOT) ...area.•Extract from HF area prolongs larval viability in vitro.•Sequential use of HF extract followed by HPOT extract support larval development.
Schistosomiasis is one of the most prevalent waterborne parasitic diseases affecting humans. In natural conditions, snails are necessary for maintenance of its lifecycle and also required as intermediate hosts to maintain the lifecycle in laboratory settings. In the present study, the location of S. mansoni larvae in Biomphalaria glabrata snails after infection (inoculation of miracidia) was investigated. Larvae were found located in the head-foot (HF) area of B. glabrata snails at 10 days post-infection (DPI), then their location was predominantly changed to the hepatopancreas and ovotestis (HPOT) area by 56 DPI. Next, the effects of extracts from various organs of B. glabrata snails including HF and HPOT for in vitro culturing of S. mansoni larvae were investigated. The HF extract enabled prolonged culturing of S. mansoni larvae. Furthermore, sequential use of that followed by the HPOT extract supported larval development or reproduction of daughter sporocysts. These results may provide important information for identifying essential factors and molecules for culturing Schistosoma larvae in vitro.
RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence ...determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.
•First report on effects of various Wnts on DP cells.•Wnt-10b promoted trichogenesis, while Wnt-3a showed to a limited extent.•Canonical Wnts, specifically Wnt-10b, is important for DP cells ...maintenance.
Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cell proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.
A comprehensive review of the infection of mammals with the nematode Dioctophyme renale (Goeze, 1782) (Nematoda, Dioctophymidae) is presented. Mammals, including man, are the definitive hosts for ...this parasite. Several aspects of the infection with the parasite in mammals other than humans are critically evaluated: geographical distribution, host species recorded so far and the relative importance of the different hosts, location of parasites within the host, prevalence and intensity of the infection, diagnostic methods, pathology induced by the parasites, epidemiology and the methods of control and treatment. The authors provide an updated review about the infection, based on a extensive bibliographic search worldwide, and point out the most relevant aspects of the biology of the parasite as well as several research topics which need to be explored for a better understanding of the biology of this interesting and important parasitic nematode.
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•Dioctophyme renale infects at least 49 different species of mammals in 33 countries.•South America followed by North America are the highest endemic Areas.•By now, no case report from Africa, Australia and most of Southeast Asian countries.•Main final host in South America is dogs, but that in North America is minks.•Natural life cycle remains unclear due to rarelity of infected animals in broad areas.
Glioblastoma is a type of intracranial malignancy. Shikonin, a Chinese traditional medicine, has been shown to have anti-tumor efficacy toward human glioblastoma cells in vitro. However, shikonin ...cannot easily cross the blood–brain barrier. To address this issue, we evaluated the anti-tumor effects of direct intracranial infusion of shikonin in in vivo orthotopic syngeneic murine glioblastoma models using C57BL/6 mice.
The cytotoxic effects of shikonin against murine glioblastoma cells, SB28 and CT-2A, were reported resistance to temozolomide, were evaluated using an allophycocyanin-conjugated annexin V and propidium iodide assay with flow cytometry. Impedance-based real-time cell analysis (RTCA) was used to analyze the inhibitory effects of shikonin on growth and proliferation. To evaluate the anti-tumor activity of shikonin in vivo, we used orthotopic syngeneic murine glioblastoma models with SB28 and CT-2A cells.
In flow cytometry-based cytotoxic assays, shikonin induced apoptosis. RTCA indicated that shikonin decreased the cell index of murine glioblastoma cells, SB28 and CT-2A, in a dose-dependent manner (p < 0.0001 for both cell lines), while temozolomide did not (p = 0.91 and 0.82, respectively). In murine glioblastoma models, SB28 and CT-2A, direct intracranial infusion of shikonin, as a local chemotherapy, improved the overall survival of mice in a dose-dependent manner compared with control groups (p < 0.0001 and p = 0.02, respectively). While temozolomide did not (p = 0.48 and 0.52, respectively).
The direct intracranial infusion of shikonin has potential as a local therapy for patients with glioblastoma.
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•Shikonin inhibited growth and induced apoptosis in glioblastoma cell lines.•Shikonin changed the cytoskeleton architecture of murine glioblastoma cells.•Murine glioblastoma cells, SB28 and CT-2A, were resistance to temozolomide.•The intracranial shikonin infusion improved the overall survival of murine models.•Treatment with shikonin resulted in improved survival time in a dose-dependent manner.
Invasive Streptococcus agalactiae (GBS) infections are increasingly common among neonates and the elderly. Therefore, GBS surveillance for better antibiotic treatment and prophylaxis strategies are ...needed. We retrospectively evaluated the clinical aspects of invasive infections and the phenotypic and genetic diversity of infectious isolates from Nara, Japan, collected between 2007 and 2016, by using information from hospital records. GBS strains collected from the blood and cerebrospinal fluid cultures were evaluated for capsular types, multi-locus sequence typing (MLST), antibiotic susceptibility, antibiotics resistance gene, and pulsed-field gel electrophoresis. Forty GBS isolates (10 from children and 30 from adults) were analyzed, and the distribution of molecular serotype and allelic profiles varied between children and adults. We found the rates of early-onset disease in neonates with birth complications to be higher than that of previous reports, indicating that there could be relevance between complications at birth and early-onset disease. Standard antibiotic prophylaxis strategies may need to be reconsidered in patients with birth complications. In adults, the mean age of the patients was 68 years (male: 63%). Primary bacteremia was the most common source of infection. In the neonates, six had early-onset diseases and four had late-onset diseases. The most frequently identified strains were molecular serotype Ia ST23 (40%) and molecular serotype Ib ST10 (20%) in children and molecular serotype Ib ST10 (17%), molecular serotype VI ST1 (13%), and molecular serotype V ST1 (13%) in adults. Levofloxacin-resistant molecular serotype Ib strains and molecular serotypes V and VI ST1 were common causes of GBS infection in adults but were rarely found in children. Furthermore, pulsed-field gel electrophoresis in our study showed that specific clone isolates, that tend to have antibiotics resistance were widespread horizontally for a decade. Continuous surveillance and molecular investigation are warranted to identify the transmission route and improve antibiotic treatment strategies.
Non-typhoidal Salmonellae are Gram negative bacilli commonly causing self-limiting gastroenteritis, representing a public health issue particularly in tropical countries. Further, the epidemiology of ...invasive infection by non-typhoidal Salmonella species is poorly understood. Herein, we presented a case of an unusual Salmonella enterica subsp. enterica serovar Altona epidural abscess that cause osteomyelitis and psoas abscess in a 52-year-old Japanese man. To ensure adequate antibiotics penetration into the epidural space, the patient was treated with antibiotics in doses similar to those administered for meningitis. We also reviewed the literature on patients who developed non-typhoidal Salmonella epidural abscesses, and we found 10 other previously reported cases. Salmonella Enteritidis was the pathogen most commonly identified, similar to gastroenteritis. More surveillance of non-typhoidal Salmonella serovars, especially in cases of severe infection, and investigation of antibiotic penetration rate into the epidural space are warranted to decide the best treatment course.
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