Identification of a de novo PUF60 variant associated with craniofacial microsomia.

Craniofacial microsomia (CFM), also known as the oculo-auriculo-vertebral spectrum, is a congenital disorder characterized by hypoplasia of the mandible and external ear due to tissue malformations originating from the first and second branchial arches. However, distinguishing it from other syndromes of branchial arch abnormalities is difficult, and causal variants remain unidentified in many cases. In this report, we performed an exome sequencing analysis of a Brazilian family with CFM. The proband was a 12-month-old boy with clinical findings consistent with the diagnostic criteria for CFM, including unilateral mandibular hypoplasia, microtia, and external auditory canal abnormalities. A heterozygous de novo nonsense variant (c.713C>G, p.S238*) in PUF60 was identified, which was predicted to be pathogenic in silico. PUF60 has been reported as a causal gene in Verheij syndrome, but not in CFM. Although the boy showed craniofacial abnormalities and developmental delay that overlapped with Verheij syndrome, the facial asymmetry with unilateral hypoplasia of the mandible observed in this case did not match the previously reported phenotypes of PUF60 variants. Our findings expand the phenotypic range of PUF60 variants that cover CFM and Verheij syndrome.

Frequent toothbrushing boosts resilience among children in poverty: results from a population-based longitudinal study.

BACKGROUND: Poverty negatively impacts beneficial aspects of mental development, such as resilience. Toothbrushing, an oral health behavior, has the potential to protect children's resilience through its anti-inflammatory and self-management effects and may be more effective for children, especially children in poverty. This study investigated whether toothbrushing boosts resilience among children, especially children under poverty, and modifies the association between poverty and resilience using a longitudinal population sample of school children. METHODS: Data from the Adachi Child Health Impact of Living Difficulty (A-CHILD Study) were analyzed. A baseline study was conducted in 2015 in which the children were in first grade and followed through fourth grade (N = 3459, response rate: 80%, follow-up rate: 82%). Poverty was assessed by material deprivation (life-related deprivation and child-related deprivation) and annual household income at baseline. Children's toothbrushing frequency was assessed at baseline and classified into less than twice a day or twice or more a day. Children's resilience was assessed at baseline and follow-up using the Children's Resilient Coping Scale (range 0-100). RESULTS: Children who brushed their teeth twice or more a day in first grade had 3.50 points greater resilience scores in fourth grade than those who brushed their teeth less than twice a day in first grade. After adjusting for confounders, including resilience in first grade, among underpoverty children, those who brushed their teeth twice or more a day in first grade had higher resilience scores [2.66 (95% CI = 0.53, 4.79)] than those who brushed their teeth less than twice a day. Among nonpoverished children, toothbrushing frequency in first grade did not significantly correlate with resilience in fourth grade. CONCLUSIONS: The beneficial effect of toothbrushing twice or more a day on resilience was more significant among children in poverty than among those without poverty in elementary school in Japan. Health policy focused on frequent toothbrushing may contribute to boosting resilience among children living in poverty.

Validation of parameters recommended for secondary screening for developmental dysplasia of the hip in Japan.

BACKGROUND: Based on the Japanese Pediatric Orthopaedic Association's guidelines, secondary screening and imaging including ultrasonography and radiography, are recommended in infants with limited hip abduction (<70°) or in those with multiple risk factors including the following: asymmetrical skin creases, a family history of developmental dysplasia of the hip, female sex, and pelvic position at delivery. However, there is still little information regarding the usefulness of this guideline. The objective of this study was to investigate the association between the risk factors and developmental dysplasia of the hip diagnosed using ultrasound and radiography. METHODS: A total of 356 infants (67 boys and 289 girls) underwent secondary ultrasonographic and radiological screening for developmental dysplasia of the hip in our hospital. Risk factors were documented from their medical records. The recommended item score, which we defined as an integrated value of the recommended item, was calculated for each patient. The limitation of hip abduction alone was a criterion for secondary screening; therefore, we defined the scores as follows: the limitation of hip abduction scored 2 points and other recommended scores were assigned 1 point. If the recommended item score was 2 points or more, we classified the infants as high-risk. RESULTS: A total of 280 of 356 infants were included in the high-risk group, which showed a higher ratio of cases with abnormal imaging findings than the low-risk group. According to the multivariate logistic regression analyses among the recommended items, being female, skin asymmetry, and limb limitation were identified as independent risk factors for imaging abnormality and the need for Pavlik harness treatment. CONCLUSIONS: The recommended items for secondary screening based on the Japanese Pediatric Orthopaedic Association's guidelines could be useful for screening infants in need of treatment.

Nasal obstruction during the growth period modulates the Wnt/ß-catenin pathway and brain-derived neurotrophic factor production in association with tyrosine kinase receptor B mRNA reduction in mouse hippocampus.

There is accumulating evidence that nasal obstruction induces high-level brain dysfunction, including memory and learning deficits. We previously demonstrated that unilateral nasal obstruction (UNO) during the growth period increases the expression of brain-derived neurotrophic factor (BDNF). The expression of BDNF is regulated by the Wnt/ß-Catenin pathway, which is linked to neuronal differentiation, proliferation, and maintenance. However, little is known about whether Wnt3a protein expression could be an index for modulations analyses in the Wnt/ß-Catenin pathway caused by UNO during the growth period. This study aimed to investigate the effects of UNO during the growth period on the Wnt/ß-Catenin pathway in the hippocampus using combined behavioural, biochemical, and histological approaches. Male BALB/C mice were randomly divided into the control (CONT; n = 6) and experimental (UNO; n = 6) groups. Blood oxygen saturation (SpO2 ) levels were measured, and a passive avoidance test was performed in mice aged 15 weeks. Brain tissues were subjected to immunohistochemistry, real-time reverse transcription-polymerase chain reaction, and western blot analysis. Compared with control mice, UNO mice had lower SpO2 levels and exhibited memory/learning impairments during behavioural testing. Moreover, Wnt3a protein, BDNF mRNA, and tyrosine kinase receptor B (TrkB) mRNA expression levels were significantly lower in the hippocampus in the UNO group than in the CONT group. Our findings suggested that UNO during the growth period appeared to modulate the hippocampal Wnt/ß-catenin pathway and BDNF production in association with TrkB mRNA reduction, thereby resulting in memory and learning impairments.

Division of the role and physiological impact of multiple lysophosphatidic acid acyltransferase paralogs.

BACKGROUND: Lysophosphatidic acid acyltransferase (LPAAT) is a phospholipid biosynthesis enzyme that introduces a particular set of fatty acids at the sn-2 position of phospholipids. Many bacteria have multiple LPAAT paralogs, and these enzymes are considered to have different fatty acid selectivities and to produce diverse phospholipids with distinct fatty acid compositions. This feature is advantageous for controlling the physicochemical properties of lipid membranes to maintain membrane integrity in response to the environment. However, it remains unclear how LPAAT paralogs are functionally differentiated and biologically significant. RESULTS: To better understand the division of roles of the LPAAT paralogs, we analyzed the functions of two LPAAT paralogs, PlsC4 and PlsC5, from the psychrotrophic bacterium Shewanella livingstonensis Ac10. As for their enzymatic function, lipid analysis of plsC4- and plsC5-inactivated mutants revealed that PlsC4 prefers iso-tridecanoic acid (C12-chain length, methyl-branched), whereas PlsC5 prefers palmitoleic acid (C16-chain length, monounsaturated). Regarding the physiological role, we found that plsC4, not plsC5, contributes to tolerance to cold stress. Using bioinformatics analysis, we demonstrated that orthologs of PlsC4/PlsC5 and their close relatives, constituting a new clade of LPAATs, are present in many γ-proteobacteria. We also found that LPAATs of this clade are phylogenetically distant from principal LPAATs, such as PlsC1 of S. livingstonensis Ac10, which are universally conserved among bacteria, suggesting the presence of functionally differentiated LPAATs in these bacteria. CONCLUSIONS: PlsC4 and PlsC5, which are LPAAT paralogs of S. livingstonensis Ac10, play different roles in phospholipid production and bacterial physiology. An enzyme belonging to PlsC4/PlsC5 subfamilies and their close relatives are present, in addition to principal LPAATs, in many γ-proteobacteria, suggesting that the division of roles is more common than previously thought. Thus, both principal LPAATs and PlsC4/PlsC5-related enzymes should be considered to decipher the metabolism and physiology of bacterial cell membranes.

Utility of thermal image scanning in screening for febrile patients in cold climates.

BACKGROUND: Infrared thermography (IRT) for fever screening systems was introduced in not only general hospitals, but also orthopedic hospitals as a countermeasure against the spread of coronavirus disease 2019 (COVID-19). Despite the widespread use of IRT, various results have shown low and high efficacies, so the utility of IRT is controversial, especially in cold climates. The aims of this study were to investigate the utility of IRT in screening for fever in a cold climate and to devise suitable fever screening in orthopedic surgery for COVID-19. METHODS: A total of 390 orthopedic surgery patients were enrolled to the outdoor group and 210 hospital staff members were enrolled to the indoor group. Thermographic temperature at the front of the face in the outdoor group was immediately measured after entering our hospital from a cold outdoor environment. Measurements for the indoor group were made after staying in the hospital (environmental temperature, 28 °C) for at least 5 h. Body temperature was then measured using an axillary thermometer >15 min later in both groups. RESULTS: In the outdoor group, mean thermographic temperature was significantly lower than axillary temperature and IRT could not detect febrile patients with axillary temperatures >37.0 °C. Mean thermographic temperature was significantly lower in the outdoor group than in the indoor group. Sensitivity was 11.5% for the outdoor group, lower than that for the indoor group. CONCLUSIONS: We verified that IRT was not accurate in a cold climate. IRT is inadequate as a screening method to accurately detect febrile individuals, so we believe that stricter countermeasures for second screening need to be employed to prevent nosocomial infections and disease clusters of COVID-19, even in orthopedic hospitals.

Radiological predictors associated with success of treatment for developmental dysplasia of hip using the Pavlik harness: A retrospective study.

BACKGROUND: While various predictors of treatment outcome of Pavlik harness have been reported, appropriate indications for treatment and relationships between patient characteristics and outcome are unclear. The present study aimed to identify radiological predictors for successful Pavlik harness treatment of DDH at the initiation of treatment and investigate the progression of radiological characteristics after successful treatment. METHODS: One-hundred-forty-two of 527 infants who visited our hospital for secondary screening of DDH were treated with the Pavlik harness. One-hundred-eight hips of 108 infants experienced successful treatment and could be followed up until 3 years of age (group S); treatment was unsuccessful within the first 2 weeks for 22 hips of 21 infants (group F). We investigated the Graf classification and radiological parameters. RESULTS: We observed the ipsilateral-side measurements of distance A and B as defined by Yamamuro and Chene's method to be significantly smaller and greater, respectively, in group F compared with those of group S. Group F tended to exhibit the smaller distance A and larger distance B compared to the group S among the same Graf type. The cut-off values for successful treatment at were >7.4 mm at initiation of treatment for distance A and <11.2 mm for distance B. The acetabular index (AI) of the ipsilateral side at the initiation of treatment was significantly correlated with that at 1 and 3 years of age. Patients with an AI of >32° at the initiation of harness treatment were more likely to exhibit hip dysplasia at 3 years of age. CONCLUSIONS: Radiographic parameters prior to the Pavlik harness could be predictors of treatment failure and DDH in the future. This study showed that AI at the initiation of treatment is associated with residual acetabular dysplasia, suggesting that radiographic assessment may be useful even considering the risks associated with radiation exposure.

Identification of OPN3 as associated with non-syndromic oligodontia in a Japanese population.

Tooth agenesis is one of the most frequent congenital abnormalities found in the maxillofacial region. Oligodontia, a severe form of tooth agenesis, occurs as an isolated anomaly or as a syndromic feature. We performed whole exome sequencing analyses to identify causative mutation in a Japanese family with three affected individuals with non-syndromic oligodontia. After variant filtering procedures and validation by Sanger sequencing, we identified one missense mutation (c.668 C > T, p.Gly223Asp) in OPN3 at 1q43, encoding a photosensitive G-protein-coupled receptor (GPCR) expressed in various tissues including brain, liver, and adipose. This mutation was predicted to be pathogenic in silico and was not found in the public databases. We further examined 48 genetically unrelated cases by targeted sequencing of the OPN3 gene region and found one additional missense variant in this gene (c.768 C > T, p.Met256Ile) that was also predicted to be pathogenic. Localization of OPN3 protein by immunohistochemical analysis using mouse embryo revealed its specific expression in the tooth gems from bud to bell stages and their surrounding tissues. These results indicated that OPN3 was involved in non-syndromic oligodontia, which has made an anchoring point for clinical application including DNA diagnostics.

Amyotrophic lateral sclerosis with speech apraxia, predominant upper motor neuron signs, and prominent iron accumulation in the frontal operculum and precentral gyrus.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease; transactivation response DNA-binding protein of 43 kDa (TDP-43) and iron accumulation are supposed to play a crucial role in the pathomechanism of the disease. Here, we report an unusual case of a patient with ALS who presented with speech apraxia as an initial symptom and upper motor neuron deficiencies. In the early clinical stages, single-photon emission computed tomography visualized focal hypoperfusion of the right frontal operculum, and magnetic resonance imaging identified a hypointense area along the frontal lobe on T2-weighted images. Neuropathological examination revealed that neuronophagia of Betz cells, gliosis, appearance of phosphorylated TDP-43 (p-TDP-43)-positive glial and neuronal inclusions, and prominent iron accumulation were frequently visible in the precentral gyrus. TDP-43 pathology and focal iron accumulation were also visible in the frontal operculum, but only a mild neuronal loss and a few p-TDP-43-positive neuronal and glial inclusions were found in the hypoglossal nucleus of the medulla oblongata and anterior horn of the spinal cord. Immunoblot analysis revealed an atypical band pattern for ALS. In our case, abnormal TDP-43 and iron accumulation might possibly have caused neurodegeneration of the frontal operculum, in tandem or independently; it might then have spread into the primary motor area. Our results suggest a causative association between TDP-43 and iron accumulation in the pathomechanisms of ALS presenting with upper motor neuron signs.

Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity.

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

Design of the N-Terminus Substituted Curvature-Sensing Peptides That Exhibit Highly Sensitive Detection Ability of Bacterial Extracellular Vesicles.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Role of acyl-CoA dehydrogenases from Shewanella livingstonensis Ac10 in docosahexaenoic acid conversion.

The biosynthesis of polyunsaturated fatty acids (PUFAs) in bacteria has been extensively studied. In contrast, studies of PUFA metabolism remain limited. Shewanella livingstonensis Ac10 is a psychrotrophic bacterium producing eicosapentaenoic acid (EPA), a long-chain ω-3 PUFA. This bacterium has the ability to convert exogenous docosahexaenoic acid (DHA) into EPA and incorporate both DHA and EPA into membrane phospholipids. Our previous studies revealed the importance of 2,4-dienoyl-CoA reductase in the conversion, suggesting that DHA is metabolized through a general ß-oxidation pathway. Herein, to gain further insight into the conversion mechanism, we analyzed the role of acyl-CoA dehydrogenase (FadE), the first committed enzyme of the ß-oxidation pathway, in DHA conversion. S. livingstonensis Ac10 has two putative FadE proteins (FadE1 and FadE2) that are highly homologous to Escherichia coli FadE. We found that FadE1 expression was induced by addition of DHA to the medium and fadE1 deletion reduced DHA conversion into EPA. Consistently, purified FadE1 exhibited dehydrogenase activity towards DHA-CoA. Moreover, its activity towards DHA- and EPA-CoAs was higher than that towards palmitoleoyl- and palmitoyl-CoAs. In contrast, fadE2 deletion did not impair DHA conversion, and purified FadE2 had higher activity towards palmitoleoyl- and palmitoyl-CoAs than towards DHA- and EPA-CoAs. These results suggest that FadE1 is the first enzyme of the ß-oxidation pathway that catalyzes DHA conversion.

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13.

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity.

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. ABBREVIATIONS: ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-ß-D-maltoside; DDM: n-dodecyl-ß-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

Long-Term Management and Maxillofacial Growth in a Klippel-Trenaunay Syndrome Patient.

Klippel-Trenaunay syndrome (KTS) is a congenital disorder associated with capillary, venous, lymphatic vascular malformations, and unilateral hypertrophy of the soft tissue and bone. We report a case of a 5-year-old girl with KTS who was followed up until age 17. The asymmetry of her maxillary dentition became remarkable with growth, although no significant left-right difference in either the maxilla or mandible was recognized. Acceptable occlusion was achieved without fixed orthodontic appliances; however, it was necessary to develop treatment plans in accordance with the general symptoms of the disease.

Effects of Zoledronate on Local and Systemic Production of IL-1ß, IL-18, and TNF-α in Mice and Augmentation by Lipopolysaccharide.

Bisphosphonates (BPs) containing nitrogen (N-BPs) exhibit far stronger anti-bone-resorptive effects than non-N-BPs. However, repeated administration of N-BPs causes osteonecrosis selectively in jawbones. As BPs accumulate in large amounts within inflamed bones, any N-BP released from the pool accumulated within jawbones might directly act on cells in the surrounding soft-tissues and induce inflammation or necrosis. Here, we examined the local and systemic effects of zoledronate (the most potent N-BP with the highest incidence of jawbone-necrosis) on inflammatory cytokines in mice. Locally within ear-pinnas: (i) zoledronate induced long-lasting accumulation of interleuikin-1ß (IL-1ß) and IL-18, but not tumor necrosis factor-α (TNF-α), (ii) zoledronate and lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria) mutually augmented the productions of IL-1ß, IL-18, and TNF-α, and (iii) oxidronate (a toxic non-N-BP) by itself produced not only IL-1ß and IL-18, but also TNF-α. In systemic experiments using intraperitoneal injection of zoledronate and/or LPS, (i) zoledronate by itself increased none of the above cytokines in serum, and (ii) in mice pretreated (3 d before) with zoledronate, the LPS-induced increases in serum IL-1ß and IL-18 were greatly augmented with a delayed slight TNF-α augmentation. These results, together with previous ones, suggest that (a) pro-IL-1ß and pro-IL-18 accumulate within cells in soft-tissues exposed to N-BPs, and infection may augment not only their production, but also the release of their mature forms, (b) IL-1ß and IL-18 (possibly together with TNF-α) may play important roles in N-BP-induced inflammation and/or necrosis, and (c) mechanisms underlying the cytotoxic effects of BPs may differ between N-BPs and non-N-BPs.

Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, Shewanella livingstonensis Ac10, as the host.

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Nuclear import of transcriptional corepressor BCOR occurs through interaction with karyopherin α expressed in human periodontal ligament.

Mutations in the gene encoding BCL-6 corepressor (BCOR) are responsible for oculofaciocardiodental (OFCD) syndrome, which is a rare X-linked dominant disorder characterized by radiculomegaly of permanent teeth as the most typical symptom. To function as a transcriptional corepressor, BCOR needs to enter the nucleus; however, the molecular pathway for its nuclear translocation during dental root formation remains unclear. The purpose of this study was to determine the mechanism underlying BCOR transport into the nucleus. Our results showed that human periodontal ligament (PDL) cells expressed karyopherin α (KPNA)2, KPNA4, and KPNA6 belonging to a family of nuclear import proteins, which interacted with BCOR in the immunoprecipitation assay. Site-directed mutagenesis targeting the two nuclear localization signals (NLSs) within BCOR reduced its nuclear translocation; however, co-expression of KPNA2, KPNA4, or KPNA6 with BCOR carrying a previously described mutation which eliminated one of the two NLSs significantly increased nuclear accumulation of the mutant BCOR, indicating participation of KPNA in BCOR nuclear translocation. Comparative expression profiling of PDL cells isolated from normal and OFCD patients revealed significant downregulation of SMAD4, GLI1, and nuclear factor 1-C (NFIC) mRNA expression, suggesting that BCOR mutations cause hyperactive root formation in OFCD syndrome by inhibiting SMAD4-Hedgehog-NFIC signaling implicated in dental root development. Our study contributes to understanding of the mechanisms providing nuclear import of BCOR during root formation.

Nasal obstruction during adolescence induces memory/learning impairments associated with BDNF/TrkB signaling pathway hypofunction and high corticosterone levels.

The hippocampus is an important brain region involved in memory and learning. Brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), and phospho-p44/p42 mitogen-activated protein kinase (MAPK) are known to contribute to hippocampal memory/learning. The present study aimed to clarify the effects of nasal obstruction during the growth period on memory/learning in an animal model, using combined behavioral, biochemical, and histological approaches. Male BALB/C mice underwent unilateral nasal obstruction (UNO) by cauterization at 8 days of age and were subjected to Y-maze and passive avoidance tests at 15 weeks of age. The serum corticosterone levels were measured using an enzyme-linked immunosorbent assay, and brain tissues were subjected to hematoxylin-eosin staining and histological analysis or homogenization and Western blot analysis. Compared with control mice, UNO mice had lower blood oxygen saturation levels and exhibited apparent memory/learning impairments during behavioral testing. Additionally, the UNO group had higher hippocampal BDNF levels and serum corticosterone levels, lower hippocampal TrkB and phospho-p44/p42 MAPK levels, and reduced neuron numbers relative to controls. Our findings suggest that UNO during adolescence affects the hippocampus and causes memory/learning impairments.