Influence of household pet ownership and filaggrin loss-of-function mutations on eczema prevalence in children: A birth cohort study.

BACKGROUND: The association between pet exposure in infancy, early childhood eczema, and FLG mutations remains unclear. METHODS: This was a birth cohort study performed in Tokyo, Japan. The primary outcome was current eczema based on questionnaire responses collected repeatedly from birth to 5 years of age. Generalized estimating equations and generalized linear modeling were used to evaluate the association. RESULTS: Data from 1448 participants were used for analyses. Household dog ownership during gestation, early infancy, and 18 months of age significantly reduced the risk of current eczema. Household cat ownership also reduced the risk of current eczema, albeit without statistical significance. The combined evaluation of children from households with pets, be it cats, dogs or both, the risk of current eczema at 1-5 years of age was lower in those with household pet exposure ownership during gestation (RR = 0.59, 95 % CI 0.45-0.77) and at 6 months (RR = 0.49, 95 % CI 0.36-0.68). , Reduced risks of eczema were also observed at 2-5 (RR = 0.52, 95 % CI 0.37-0.73) and 3-5 years of age (RR = 0.50 95 % CI 0.35-0.74) when the respective household pet ownership were evaluated at 18 months and 3 years of age. These protective associations of reduced risk of eczema were only observed in children without FLG mutations. CONCLUSIONS: Household dog and pet (dog, cat, or both) ownership was protective against early childhood eczema in a birth cohort dataset. This protective association was observed only in children without FLG mutations, which should be confirmed in studies with larger cohorts.

[A CASE OF INDUCIBLE LARYNGEAL OBSTRUCTION UNSUCCESSFULLY TREATED AS SEVERE ASTHMA WAS WELL CONTROLLED AFTER APPROPRIATE DIAGNOSIS AND INSTRUCTION OF THERAPEUTIC BREATHING MANEUVERS].

BACKGROUND: Inducible laryngeal obstruction (ILO) refers to respiratory disorders caused by airflow limitation in the larynx, including vocal cord dysfunction, and may sometimes be misdiagnosed as bronchial asthma (BA). Here, we report the case of an 11-year-old boy diagnosed with BA in infancy. He was referred to our Allergy Center and was taking a high dose of inhaled corticosteroids (ICS) due to frequent coughing from the age of 10 years and persistent coughing following COVID-19 infection at the age of 11. However, the patient continued to experience frequent coughing attacks and repeated visits to the emergency department after inhalation of ß2-stimulants failed to improve his cough. We admitted him to the allergy center for examinations to assess the BA severity. In the airway hypersensitiveness test, saline inhalation performed prior to methacholine inhalation caused expiratory stridor and respiratory distress in the larynx, which worsened with ß2-stimulant inhalation. Based on these results, we ruled out BA and diagnosed ILO. We instructed him on breathing maneuvers, and he was able to respond appropriately when symptoms appeared. We then started reducing his ICS dose.

PLPBP mutations cause variable phenotypes of developmental and epileptic encephalopathy.

OBJECTIVE: Vitamin B6-dependent epilepsies are treatable disorders caused by variants in several genes, such as ALDH7A1,PNPO, and others. Recently, biallelic variants in PLPBP, formerly known as PROSC, were identified as a novel cause of vitamin B6-dependent epilepsies. Our objective was to further delineate the phenotype of PLPBP mutation. METHODS: We identified 4 unrelated patients harboring a total of 4 variants in PLPBP, including 3 novel variants, in a cohort of 700 patients with developmental and epileptic encephalopathies. Clinical information in each case was collected. RESULTS: Each patient had a different clinical course of epilepsy, with seizure onset from the first day of life to 3 months of age. Generalized tonic-clonic seizures were commonly noted. Myoclonic seizures or focal seizures were also observed in 2 patients. Interictal electroencephalography showed variable findings, such as suppression burst, focal or multifocal discharges, and diffuse slow activity. Unlike previous reports, all the patients had some degree of intellectual disability, although some of them had received early treatment with vitamin B6, suggesting that different mutation types influence the severity and outcome of the seizures. SIGNIFICANCE: PLPBP variants should be regarded as among the causative genes of developmental and epileptic encephalopathy, even when it occurs after the neonatal period. Early diagnosis and proper treatment with pyridoxine or pyridoxal phosphate is essential to improve the neurologic prognosis in neonates or young children with poorly controlled seizures.

Insufficient sleep syndrome: An unrecognized but important clinical entity.

BACKGROUND: A sleep clinic for adults and children was established in the Tokyo Bay Urayasu Ichikawa Medical Centre, in August 2012. Given that few sleep clinics are available in Japan specifically for children, this clinic provides the opportunity to provide data on child patients with sleep problems. METHODS: Records of patients who visited the sleep clinic at the Tokyo Bay Urayasu Ichikawa Medical Centre aged ≤20 years at the first visit were retrospectively examined, along with the initial and final diagnoses. RESULTS: Of 2,157 patients who visited the sleep clinic at Tokyo Bay Urayasu Ichikawa Medical Centre between August 2012 and March 2017, 181 were ≤20 years old. In these 181 patients, the most frequent final diagnosis was insufficient sleep syndrome (ISS), n = 56, followed by circadian rhythm sleep-wake disorder, n = 28; insomnia, n = 28; and sleep-related movement disorder, n = 15. CONCLUSIONS: Insufficient sleep produces various brain dysfunctions in both adults and children, and is associated with behavioral, cognitive and physical problems, as well as with atypical early development. Insufficient sleep has also been reported to cause obesity. Insufficient sleep-induced obesity is often associated with the occurrence of metabolic syndrome. More effort is needed to ensure that children are receiving sufficient sleep.

Molecular mechanism of trans-translation.

tmRNA has a dual function as a tRNA and an mRNA to relieve the stalled ribosome. During this process, tmRNA enters the ribosomal A-site without a codonanticodon interaction, but with a protein factor SmpB. Here, we established in vitro trans-translation system, which is able to evaluate peptidyl transfer to alanyltmRNA and translation of the resume codon on tmRNA. Using this system, the effects of mutations on tmRNA or SmpB are assessed by measuring the incorporations of labeled amino acid into polypeptide. Interaction of SmpB with ribosome and tmRNA was studied by directed hydroxyl radical probing. Our results revealed that SmpB constitutes an important element during trans-translation. We propose a new model of transtranslation.

Thermus thermophilus tmRNA and trans-translation.

In trans-translation, ribosome switches the template for protein synthesis from an mRNA to tmRNA. The molecular mechanism of trans-translation remains mysterious. In order to clarify how tmRNA move through the ribosome, we developed in vitro systems to monitor trans-translation as well as translation, which are composed of ribosome, elongation factors, tmRNA and SmpB all from Thermus thermophilus. In these systems, the early steps of trans-translation including trans-transfer and the resume codon decoding could be monitored. Using these systems, the function of ribosomal protein S1 which has been suggested to be involved in trans-translation was investigated.

A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation.

In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by -1. We also found that U(85) is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U(85) from chemical modification.

In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation.

Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.

Competition between trans-translation and termination or elongation of translation.

The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3'-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3'-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.

Functional analysis of factors involved in trans-translation.

trans-translation is a rescue system for stalled ribosomes to terminate the translation for recycling of ribosome and lead the incomplete protein on the stalled ribosome to degradation. In trans-translation, several proteins and RNAs are involved. tmRNA and SmpB are only factors known to be essential for trans-translation. EF-Tu and EF-G functions in translation and also are seemed to function in trans-translation. Ribosomal protein S1 is known to bind tmRNA. In this study, we cloned the gene of tmRNA from Thermus thermophilus and analyzed the functions of factors involved in trans-translation in vitro.

In vitro analysis of the initial steps of trans-translation.

tmRNA has a dual function both as tRNA and mRNA and facilitates trans-translation. We observed the tagging derived from tmRNA from Bacillus subtilis in vivo and in vitro. Our studies give progressive suggestions on the mechanism of trans-translation.

Trans-translation mediated by Bacillus subtilis tmRNA.

Trans-translation, in which a ribosome switches between translation of an mRNA and a tmRNA, produces a chimera polypeptide of an N-terminal truncated polypeptide and a C-terminal tag-peptide encoded by tmRNA. One of the tmRNA binding proteins, a ribosomal protein S1, has not been found in a group of Gram-positive bacteria. In this study, the trans-translation reaction with tmRNA from Bacillus subtilis belonging to this group was examined. When a truncated gene lacking a termination codon was expressed in B. subtilis, a 15-amino acid tag-peptide derived from tmRNA was identified in the C-termini of the trans-translation products. An identical tag-peptide was also found at the C-termini of the products from a truncated gene, when it was coexpressed with B. subtilis tmRNA in Escherichia coli. B. subtilis tmRNA was functional, although much less efficiently, in the in vitro poly(U)-dependent tag-peptide synthesis system of E. coli. A comparison of two bacterial tmRNAs suggests that the rule for determining the tag-initiation point on tmRNA may be the same in Gram-positive and Gram-negative bacteria.

The structure and function of tmRNA.

tmRNA has a dual function both as tRNA and mRNA and facilitates trans-translation. We made various mutation in tRNA domain of tmRNA. Our mutation studies give progressive suggestions on the structure of tmRNA.