The identified clinical features of Parkinson's disease in homo-, heterozygous and digenic variants of PINK1.

To investigate the prevalence and genotype-phenotype correlations of phosphatase and tensin homolog induced putative kinase 1 (PINK1) variants in Parkinson's disease (PD) patients, we analyzed 1700 patients (842 familial PD and 858 sporadic PD patients from Japanese origin). We screened the entire exon and exon-intron boundaries of PINK1 using Sanger sequencing and target sequencing by Ion torrent system. We identified 30 patients with heterozygous variants, 3 with homozygous variants, and 3 with digenic variants of PINK1-PRKN. Patients with homozygous variants presented a significantly younger age at onset than those with heterozygous variants. The allele frequency of heterozygous variants in patients with age at onset at 50 years and younger with familial PD and sporadic PD showed no differences. [123I]meta-iodobenzylguanidine (MIBG) myocardial scintigraphy indicated that half of patients harboring PINK1 heterozygous variants showed a decreased heart to mediastinum ratio (12/23). Our findings emphasize the importance of PINK1 variants for the onset of PD in patients with age at onset at 50 years and younger and the broad spectrum of clinical symptoms in patients with PINK1 variants.

Suitable methods of measuring acceleration time in the diagnosis of internal carotid artery stenosis.

PURPOSE: To enhance the utility of acceleration time (AcT) in the diagnosis of internal carotid artery (ICA) stenosis, we assessed the value of AcT measurements with different waveform patterns. METHODS: Ninety-three patients with acute atherothrombotic cerebral infarction were enrolled, and they underwent both carotid ultrasonography and digital subtraction angiography (DSA). AcT was determined by a conventional procedure (using the first peak point or the bending point) and the peak systolic velocity (PSV) procedure. The AcT ratio was calculated as (AcT of ICA)/(AcT of the ipsilateral common carotid artery). We evaluated the correlation of stenosis rate as assessed by the North American Symptomatic Carotid Endarterectomy Trial method using DSA (DSA-NASCET) with the AcT of ICA (ICA-AcT), the AcT ratio measured by the conventional procedure (conventional AcT ratio), and the AcT ratio measured by the PSV procedure (PSV AcT ratio). The area under receiver operating characteristic curves (AUC) for DSA-NASCET was calculated based on the ICA-AcT and AcT ratio. RESULTS: Forty-five vessels had 50% or greater ICA stenosis. DSA-NASCET was positively correlated with the conventional AcT ratio (r = 0.723), conventional ICA-AcT (r = 0.638), and PSV AcT ratio (r = 0.245). The corresponding AUCs for ICA stenosis ≥ 50% were 0.971, 0.886, and 0.572, respectively. CONCLUSION: We demonstrated the usefulness of the conventional procedure for diagnosing stenosis of ICA origin using AcT and showed that the AcT ratio was a more beneficial parameter than AcT.

Dissipation, dehalogenation, and denitration of chloroaromatic compounds by Nocardioides sp. strain PD653: Characterization of the substrate specificity.

The substrate range of Nocardioides sp. strain PD653, capable of mineralizing hexachlorobenzene, was investigated based on the dissipation of substrates and the liberation of halogen ions. Strain PD653 dehalogenated 10 out of 18 halophenol congeners; however, it could dehalogenate only hexachlorobenzene out of seven halobenzene congeners tested. Moreover, dehalogenation activities were shown for chloronitrobenzenes, along with an increase in the number of substituted chlorine atoms except for 2,3,4,5-tetrachloro-1-nitrobenzene. These results suggested that this strain might be applicable to remediate soil contaminated with these persistent chloroaromatic compounds.

Identification of the novel hcbB operon catalyzing the dechlorination of pentachlorophenol in the Gram-positive bacterium Nocardioides sp. strain PD653.

While pcp genes are well known in Gram-negative bacteria to code for the enzymes responsible for pentachlorophenol (C6HCl5O; PCP) degradation, little is known about PCP-degrading genes in Gram-positive bacteria. Here we describe a novel gene operon possibly responsible for catalyzing the degradation of PCP in the Gram-positive bacterium Nocardioides sp. strain PD653, which is capable of mineralizing hexachlorobenzene (C6Cl6; HCB) via PCP. Transcriptome analysis based on RNA-Seq revealed overexpressed genes in strain PD653 following exposure to HCB. Based on in silico annotation, three open reading frames (ORFs) were selected as biodegrading enzyme candidates. Recombinant E. coli cells expressing candidate genes degraded approximately 9.4 µmol L-1 PCP in 2 hr. Therefore, we designated these genes as hcbB1, hcbB2, and hcbB3. Interestingly, PCP-degrading activity was recorded when hcbB3 was coexpressed with hcbB1 or hcbB2, and the function of HcbB3 was expected to be similar to chlorophenol 4-monooxygenase (TftD).

Identification and functional characterization of novel mutations including frameshift mutation in exon 4 of CSF1R in patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia.

OBJECTIVE: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is caused by mutations in CSF1R. Pathogenic mutations in exons 12-22 including coding sequence of the tyrosine kinase domain (TKD) of CSF1R were previously identified. We aimed to identify CSF1R mutations in patients who were clinically suspected of having ALSP and to determine the pathogenicity of novel CSF1R variants. METHODS: Sixty-one patients who fulfilled the diagnostic criteria of ALSP were included in this study. Genetic analysis of CSF1R was performed for all the coding exons. The haploinsufficiency of CSF1R was examined for frameshift mutations by RT-PCR. Ligand-dependent autophosphorylation of CSF1R was examined in cells expressing CSF1R mutants. RESULTS: We identified ten variants in CSF1R including two novel frameshift, five novel missense, and two known missense mutations as well as one known missense variant. Eight mutations were located in TKD. One frameshift mutation (p.Pro104LeufsTer8) and one missense variant (p.His362Arg) were located in the extracellular domain. RT-PCR analysis revealed that the frameshift mutation of p.Pro104LeufsTer8 caused nonsense-mediated mRNA decay. Functional assay revealed that none of the mutations within TKD showed autophosphorylation of CSF1R. The p.His362Arg variant located in the extracellular domain showed comparable autophosphorylation of CSF1R to the wild type, suggesting that this variant is not likely pathogenic. CONCLUSIONS: The detection of the CSF1R mutation outside of the region-encoding TKD may extend the genetic spectrum of ALSP with CSF1R mutations. Mutational analysis of all the coding exons of CSF1R should be considered for patients clinically suspected of having ALSP.

Usefulness of acceleration time ratio in diagnosis of internal carotid artery origin stenosis.

PURPOSE: The acceleration time (AcT) ratio of the internal carotid artery (ICA) is increased in ICA stenosis. However, there are few reports that have directly compared the AcT ratio to digital subtraction angiography (DSA) findings. METHODS: We evaluated 177 vessels with DSA and carotid artery ultrasonography. The AcT ratio was calculated as AcT of the ICA (ICA-AcT)/AcT of the ipsilateral common carotid artery (CCA). We evaluated the correlation of DSA-NASCET stenosis with the origin of the ICA or the peak systolic velocity (ICApsv) in the stenotic region, ICApsv/peak systolic velocity of the CCA (CCApsv), ICA-AcT, and AcT ratio. Sensitivity and specificity for stenosis ≥ 70% were calculated based on the ICApsv, ICApsv/CCApsv, ICA-AcT, and AcT ratio. RESULTS: Using NASCET criteria, 34 vessels had 70% or greater stenosis. DSA-NASCET showed a significant positive correlation with ICApsv, ICApsv/CCApsv, ICA-AcT, and AcT ratio (p < 0.0001). When the cut-off value for ICApsv was set at 176 cm/s, ICApsv/CCApsv at 2.42, ICA-AcT at 0.095 s, and the AcT ratio at 1.35, the sensitivity was 97.1, 97.1, 82.4, and 97.1%, and the specificity was 94.4, 91.0, 83.2, and 83.2%, for DSA-NASCET ≥ 70%, respectively. CONCLUSION: The AcT ratio is a beneficial parameter for evaluating ICA stenosis as well as ICApsv and ICApsv/CCApsv.

Identification of the hcb Gene Operon Involved in Catalyzing Aerobic Hexachlorobenzene Dechlorination in Nocardioides sp. Strain PD653.

Nocardioides sp. strain PD653 was the first identified aerobic bacterium capable of mineralizing hexachlorobenzene (HCB). In this study, strain PD653-B2, which was unexpectedly isolated from a subculture of strain PD653, was found to lack the ability to transform HCB or pentachloronitrobenzene into pentachlorophenol. Comparative genome analysis of the two strains revealed that genetic rearrangement had occurred in strain PD653-B2, with a genomic region present in strain PD653 being deleted. In silico analysis allowed three open reading frames within this region to be identified as candidate genes involved in HCB dechlorination. Assays using recombinant Escherichia coli cells revealed that an operon is responsible for both oxidative HCB dechlorination and pentachloronitrobenzene denitration. The metabolite pentachlorophenol was detected in the cultures produced in the E. coli assays. Significantly less HCB-degrading activity occurred in assays under oxygen-limited conditions ([O2] < 0.5 mg liter-1) than under aerobic assays, suggesting that monooxygenase is involved in the reaction. In this operon, hcbA1 was found to encode a monooxygenase involved in HCB dechlorination. This monooxygenase may form a complex with the flavin reductase encoded by hcbA3, increasing the HCB-degrading activity of PD653.IMPORTANCE The organochlorine fungicide HCB is widely distributed in the environment. Bioremediation can effectively remove HCB from contaminated sites, but HCB-degrading microorganisms have been isolated in few studies and the genes involved in HCB degradation have not been identified. In this study, possible genes involved in the initial step of the mineralization of HCB by Nocardioides sp. strain PD653 were identified. The results improve our understanding of the protein families involved in the dechlorination of HCB to give pentachlorophenol.

Prevalence of Right to Left Shunts in Japanese Patients with Migraine: A Single-center Study.

Objective Several studies have shown an increased prevalence of right-to-left shunt (RLs) in migraine patients, particularly those with aura. However, the prevalence of RLs and its relation to Japanese patients with migraine are unknown. We investigated the prevalence of RLs in Japanese patients with migraine. Methods In total, 112 consecutive patients with migraine were recruited from our headache outpatient clinic. Migraine with aura (MA) and migraine without aura (MWOA) were diagnosed according to the International Classification of Headache Disorders, 3rd edition (beta-version). Contrast transcranial Doppler ultrasound was used to detect RLs, including patent foramen ovale (PFO). Then, the associations between RLs and patients' backgrounds and presence of aura were assessed. Results The overall prevalence of RLs and PFO in migraine patients was 54.5% and 43.8%, respectively. The prevalence of RLs and PFO in the MA group were significantly higher than in the MWOA group (RLs, 62.9% vs. 44.0%, p=0.046; PFO, 54.8% vs. 30.0%, p=0.008). There were no marked differences in the prevalence of large, middle and small shunts between MA and MWOA patients. Compared with the MWOA patients, the MA patients were younger (p=0.013) and had early onset age (p=0.013) and increased prevalence of photophobia (p=0.008). Conclusion RLs were found in over half of the Japanese patients with migraine. Our study suggests a possible link between RLs and MA.

Human herpesvirus 6 encephalitis followed by acute disseminated encephalomyelitis in an immunocompetent adult.

A 26-year-old, otherwise healthy man presented with visual abnormality followed by loss of consciousness and convulsion. The patient then developed headache and fever 14 days later. Brain MRI showed hyperintensities in the left cingulate cortex. The cerrebrospinal fluid examinations showed mononuclear pleocytosis and positive PCR results for human herpesvirus 6 (HHV-6). A diagnosis of HHV-6 encephalitis and symptomatic epilepsy was made. The patient's clinical symptoms improved promptly following acyclovir treatment. However, 3 months later the patient noticed dysesthesia in the trunk, the left upper limb and the right lower limb. Brain and spine MRI showed multiple brain white matter lesions, the middle cerebellar peduncle and cervical spinal lesions. The symptoms resolved following methylprednisolone pulse therapy only. We report an adult patient with HHV-6 encephalitis followed by acute disseminated encephalomyelitis whose initial presentation was epilepsy. HHV-6 encephalitis should be included in the differential diagnosis of encephalitis of unknown etiology in an immunocompetent adult.

Increased maximum common carotid intima-media thickness is associated with smoking and hypertension in Tochigi Prefecture residents.

OBJECTIVE: We investigated maximum intima-media thickness of the common carotid artery (IMT-Cmax) in residents of Tochigi Prefecture, who have been reported to have high stroke mortality. METHOD: Our study included 840 individuals. All participants underwent carotid ultrasonography and answered a questionnaire during participation in a health festival in Tochigi Prefecture. The questionnaire was designed to collect information on age, gender, and risk factors for stroke. IMT-Cmax was measured. Statistical analyses were performed to identify factors contributing to IMT-Cmax values ≥1.1 mm. RESULTS: In total, 117 subjects had an IMT-Cmax value ≥1.1 mm. IMT-Cmax correlated significantly with age, current smoking, hypertension, diabetes mellitus, heart disease, and previous symptomatic stroke (p < 0.05) in univariate analysis. Current smoking (p < 0.001, odds ratio 3.88) and hypertension (p = 0.0070, odds ratio 1.83) were seen as significant contributing factors to IMT-Cmax ≥1.1 mm in logistic regression analysis adjusted by age, gender, and previous symptomatic stroke. CONCLUSION: We identified current smoking and hypertension as the most significant contributing factors to increased IMT-Cmax in residents of Tochigi Prefecture, emphasizing the importance of routine blood pressure monitoring and anti-smoking education in this population.

The relationship between right-to-left shunt and brain white matter lesions in Japanese patients with migraine: a single center study.

BACKGROUND: There may be a link between right-to-left shunt (RLs) and brain white matter lesions (WMLs) in patients with migraine. In this study, we assessed the relationship between WMLs and RLs in Japanese migraine patients. METHODS: A total of 107 consecutive patients with migraine with (MA) and without aura (MWOA) were included in this study. Contrast transcranial Doppler ultrasound was used to detect RLs. WMLs were graded using brain magnetic resonance imaging based on well-established criteria. FINDINGS: The prevalence of RLs was significantly increased in the WMLs positive group (n = 24) compared with the WMLs negative group (n = 83) (75.0% vs. 47.0%, p = 0.015). In prevalence of WMLs between MA and MWOA patients, there were no statistical differences (p = 0.410). Logistic regression analysis adjusted by age and disease duration of migraine identified an RLs-positive status as the sole determinant for the presence of WMLs (OR = 6.15; 95% CI 1.82-20.8; p = 0.003) CONCLUSION: Our study suggests a possible link between RLs and WMLs in Japanese patients with migraine.

Production of secretory cutinase by recombinant Saccharomyces cerevisiae protoplasts.

BACKGROUND: During heterologous protein production using recombinant microbes, the protein tends to accumulate in the cell and may not be secreted. Here, we studied the production of secretory cutinase (heterologous protein) by recombinant Saccharomyces cerevisiae protoplasts. FINDINGS: Recombinant S. cerevisiae cells (i.e., cells into which the cutinase gene was transferred) secreted trace amounts of cutinase into the broth. Approximately 28 % of the cutinase produced in the cells localized to the cell walls and/or between cell wall and cell membrane (CW). In comparison with cell culture, protoplasts in a static culture secreted measurable amounts of cutinase into the broth. Protoplasts were protected from physical and osmotic stresses by entrapping them in a membrane capsule with a low-viscous liquid-core of 1.92 % w/v calcium-alginate. To increase secretory cutinase production, the entrapped protoplasts were cultivated in a shake flask at low osmotic pressure without disruption. During 60 h of cultivation, the extracellular cutinase activity of the free protoplasts at 29.3 atm and protoplasts entrapped in the capsule at 17.2 atm were 0.13 and 0.39 U/mL, respectively. CONCLUSIONS: This is the first report which demonstrates that the efficient production of a secretable enzyme by using protoplasts isolated from recombinant microbes. This system described here is useful to produce products that accumulate in the CW.

Recurrent juvenile ischemic stroke caused by bow hunter's stroke revealed by carotid duplex ultrasonography.

Bow hunter's stroke (BHS) is a rare cause of vertebrobasilar insufficiency due to rotational vertebral artery (VA) occlusion associated with head turning. We report a juvenile patient presenting with recurrent ischemic stroke caused by BHS, which was revealed by carotid duplex ultrasonography. Carotid duplex ultrasonography performed in the neutral position showed normal findings. However, disappearance of end-diastolic blood flow of contralateral VAs was observed with head rotation. Digital subtraction angiography confirmed occlusion at C1/2 levels in the VA contralateral to the head rotation, bilaterally. Importantly, our patient did not recognize the association of head rotation and previous episodes of stroke. We suggest that BHS should be considered in patients with cryptogenic stroke occurring in the vertebrobasilar artery territory.

Post-irradiation Skin Changes Associated with Lumbosacral Radiculopathy.

We herein describe the cases of two patients with post-irradiation lumbosacral radiculopathy. The patients underwent postoperative radiation therapy to the abdomen due to testicular neoplasms 20 and 25 years prior to the onset of weakness, respectively. On physical examinations, asymmetric lower limb weakness and areflexia without apparent sensory loss were observed in both patients. Interestingly, artificial and squared atrophy of the skin and subcutaneous tissue, thought to correspond to the radiation fields, were observed in the lower back, and electromyography revealed selective motor axon loss localized to the lower extremities. The detection of skin changes in the area being irradiated is a valuable clue for diagnosing post-irradiation lumbosacral radiculopathy.

Sleep apnoea headache in obstructive sleep apnoea syndrome patients presenting with morning headache: comparison of the ICHD-2 and ICHD-3 beta criteria.

BACKGROUND: Morning headache is associated with obstructive sleep apnoea syndrome (OSAS); however, OSAS patients present with various characteristics of morning headache, and they often do not fulfil the International Classification of Headache Disorders (ICHD)-2 criteria for "sleep apnoea headache". The aims of this study were to assess the new ICHD-3 beta criteria for sleep apnoea headache in OSAS patients and to evaluate the differences with the ICHD-2. METHODS: We conducted a cross-sectional survey regarding morning and sleep apnoea headaches that included 235 OSAS outpatients receiving continuous positive airway pressure (CPAP) treatment. The presence of morning headache was evaluated by reviewing the medical records before administration of CPAP treatment. RESULTS: Of all of the OSAS patients, 48 (20.4%) reported morning headaches. Of the 48 patients with morning headaches, 29 (60.4%) and 39 (81.3%) fulfilled the ICHD-2 and ICHD-3 beta criteria for sleep apnoea headache, respectively. The increased frequency of individuals who qualified for diagnosis was likely attributable to the extension of headache duration from 30 min to 4 h. The severity of OSAS was not associated with the presence of sleep apnoea headache. CONCLUSIONS: The utilisation of ICHD-3 beta criteria is clinically useful for diagnosing sleep apnoea headache in patients with OSAS. Applying the ICHD-3 beta criteria was of clinical significance when considering the marked response of these headaches to CPAP therapy. The cause of sleep apnoea headache remains to be elucidated.

[Lateral medullary infarction presenting with truncal lateropulsion and decreased pain and temperature sensation below T10 sensory level].

A 45-year-old man noted a tendency to fall to the left side on standing following headache and vomiting. Neurological findings were unremarkable except for truncal lateropulsion to the left side. Brain magnetic resonance imaging (MRI) showed a small infarct in the left inferolateral part of medulla. Clinical and imaging findings suggested vertebral artery dissection as the cause of stroke. On hospital day 6, the patient developed decreased pain and temperature sensation in the right side below T10 sensory level and decreased sweating on the left side of the face with miosis of the left pupil. Brain MRI revealed an expanded lesion of the left inferolateral part of medulla. In our patient, the involvement of the spinocerebellar tract and the lateral spinothalamic tract likely contributed to the development of truncal lateropulsion and sensory disturbance below thoracic levels, respectively. Our case is clinically important in understanding a correlation between clinical symptoms and lateral medullary lesions.

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species.

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.

Spectrophotometric detection of labile zinc(II) released from metallothionein: a simple method to evaluate heavy metal toxicity.

A new and simple method based on the mechanism of detoxification of metallothionein was developed by using a water-soluble porphyrin and Zn(II)-bound metallothionein for evaluating heavy metal toxicity. Labile Zn(II) ions were released when toxic metal ions such as Cu(II), Pb(II), Bi(III), Cd(II), Hg(II), Co(II), Ag(I), and Ni(II) bound to Zn(II)-bound metallothionein. The water-soluble porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphinetetrasulfonic acid, a chromogenic reagent that is highly sensitive to Zn(II), formed a complex with the labile Zn(II) ions. The absorption change at 423 nm resulting from the formation of the Zn(II)-porphyrin complex was used to evaluate the toxicity of sample solutions containing different metal ions. The absorption change was well correlated with the toxicity, which was evaluated by a bioluminescence inhibition assay using the bioluminescent bacteria Vibrio fischeri. This observation indicated that the absorption change determined by our method was a good indicator of heavy metal toxicity. The proposed method was more sensitive than conventional bioassays and could be used to detect metal toxicity at submicromolar concentrations of toxic metal ions.