Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation.

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

Carbonylated Proteins as Key Regulators in the Progression of Metabolic Syndrome.

Based on the known role of oxidative stress in the pathogenesis and progression of metabolic syndrome, we used two-dimensional gel electrophoresis with immunochemical detection of protein carbonyls (2D-Oxyblot) to characterize the carbonylated proteins induced by oxidative stress in spontaneously hypertensive rats/NDmcr-cp (CP), an animal model of metabolic syndrome. We also profiled the proteins that showed change of expression levels in their epididymal adipose tissue at the pre-symptomatic (6-week-old) and the symptomatic (25-week-old) stages of the metabolic syndrome. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to analyze proteins extracted from the epididymal adipose tissue. The up-regulated proteins identified at the pre-symptomatic stage were mainly associated with ATP production and redox reaction, while the down-regulated proteins found at the symptomatic stage were involved in antioxidant activity and the tricarboxylic acid (TCA) cycle. Further analysis using the 2D-Oxyblot showed significantly high carbonylation levels of gelsolin and glycerol-3-phosphate dehydrogenase [NAD+] at the symptomatic stage. These results suggest that reduced antioxidant capacity underlies the increased oxidative stress state in the metabolic syndrome. The identified carbonylated proteins, including gelsolin, are potential targets that may act as key regulators in the progression of the metabolic syndrome.

Association of activities of daily living and cognitive function with thickness of the upper extremity muscles in children and adults with cerebral palsy.

PURPOSE: We examined the association of activities of daily living (ADL) and cognitive function with the upper extremity muscle thickness and upper extremity range of motion (ROM) and spasticity in children and adults with cerebral palsy (CP). METHODS: The subjects were 20 children and adults with CP. The ADL performed using the upper extremities and cognitive function were assessed using the self-care domain of the Pediatric Evaluation of Disability Inventory (PEDI) and the full-scale intelligence quotient (FSIQ) of the Wechsler Intelligence Scale for Children, fourth edition (WISC-IV), respectively. The WISC-IV was assessed in only seven of 20 subjects able to undergo evaluation. The thickness of the upper extremity muscles was measured using an ultrasound imaging device. Moreover, ROM and spasticity of the upper extremities were assessed using the Modified Ashworth Scale (MAS). Manual manipulation ability was also assessed using the Manual Ability Classification System (MACS). RESULTS: Stepwise regression analysis revealed that the extensor digitorum muscle thickness and MACS level were significant and independent factors of self-care in the PEDI. Partial correlation analysis with MACS level and age as control variables showed that the FSIQ of the WISC-IV was significantly associated with the thickness of the anterior fibers of the deltoid and flexor digitorum superficialis muscles. CONCLUSION: Reduced ADL performed using the upper extremities is associated with decreased extensor digitorum muscle thickness rather than ROM and spasticity of the upper extremities in children and adults with CP.

In Vitro Exposure to Glucose Alters the Expression of Phosphorylated Proteins in Platelets.

Diabetes mellitus (DM) is a pro-thrombotic state that can potentially cause serious cardiovascular complications. Platelet hyperactivation plays an important role in these pathological processes, however there is little or no information on the effect of hyperglycemia on platelet proteins. The aim of this study was to identify the molecular targets associated with platelet reactivity under hyperglycemia. Towards this goal, we examined the effects of the exposure of platelets to 1 and 2 h glucose (300 mg/dL) and control (vehicle and osmolality control using mannitol) on platelet proteins (n = 4 samples per group) using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem mass spectrometry. Two-hour exposure to glucose significantly up-regulated the expression of ATP synthase subunit beta, filamin-A, and L-lactate dehydrogenase A chain in platelets. Pro-Q Diamond staining confirmed the effect of 2 h glucose on vinculin, heat shock protein HSP 90-alpha, filamin-A, and fructose-bisphosphate aldolase A (platelet phosphorylated proteins). The identified proteins are involved in various cellular processes and functions and possibly in platelet reactivity under hyperglycemic conditions.

Pleasant Stroke Touch on Human Back by a Human and a Robot.

Pleasant touching is an important aspect of social interactions that is widely used as a caregiving technique. To address the problems resulting from a lack of available human caregivers, previous research has attempted to develop robots that can perform this kind of pleasant touch. However, it remains unclear whether robots can provide such a pleasant touch in a manner similar to humans. To investigate this issue, we compared the effect of the speed of gentle strokes on the back between human and robot agents on the emotional responses of human participants (n = 28). A robot or a human stroked on the participants' back at two different speeds (i.e., 2.6 and 8.5 cm/s). The participants' subjective (valence and arousal ratings) and physiological (facial electromyography (EMG) recorded from the corrugator supercilii and zygomatic major muscles and skin conductance response) emotional reactions were measured. The subjective ratings demonstrated that the speed of 8.5 cm/s was more pleasant and arousing than the speed of 2.6 cm/s for both human and robot strokes. The corrugator supercilii EMG showed that the speed of 8.5 cm/s resulted in reduced activity in response to both human and robot strokes. These results demonstrate similar speed-dependent modulations of stroke on subjective and physiological positive emotional responses across human and robot agents and suggest that robots can provide a pleasant touch similar to that of humans.

Potential Contribution of Ultrasonography Assistance for the Safe and Steady Procedure of Endoscopic Intracerebral Hematoma Evacuation: A Retrospective Cohort Study.

BACKGROUND: Endoscopic hematoma evacuation is one of the most promising procedures for the treatment of intracerebral hemorrhage (ICH) to avoid severe outcomes, such as death or dependency. However, the effect of the procedure on the functional outcome remains controversial. Thus, standardization and sophistication are required to enhance the surgical results. This study aimed to evaluate the potential efficacy of ultrasonography (US) in endoscopic hematoma evacuation. METHODS: This study included 39 consecutive patients with spontaneous supratentorial ICH who underwent endoscopic hematoma evacuation between April 2019 and July 2021. The patients were divided into two groups, namely, surgery with or without US assistance. Rebleeding and evacuation rate were set as the primary endpoints, and operation time, requirement for repeat puncture, and modified Rankin scale at discharge were set as the secondary endpoints. During surgery, the burr hole was placed, and the dura mater was widely opened. The US probe was applied on the brain surface via the burr hole to detect the depth and direction of the hematoma cavity. With US assistance, the hematoma cavity was punctured with a cannula, and the transparent port was introduced into the hematoma cavity along the tract. The hematoma was gently evacuated with the irrigation-suction instrument. RESULTS: Of the 39 cases, 9 underwent endoscopic hematoma evacuation with US assistance. Rebleeding was noted in 0 and 2 (6.7%) patients with and without US assistance, respectively (p = 0.43). The mean hematoma evacuation rates were 78.6 and 80.6% in patients with and without US assistance, respectively (p = 0.80). In all cases with US assistance, the cavity could be reached with a single tap. However, repeat puncture was required in 20 (66.7%) cases without US assistance (p = 0.04). In one case, an unexpected residual hematoma was detected using US, which was applied after hematoma evacuation and before wound closure. The operation time was not extended even if US was used during the surgery. CONCLUSIONS: US-assisted hematoma evacuation is an effective procedure that can assist in the precise insertion of the puncture cannula and exclusion of the residual hematoma. US might contribute toward improving the accuracy of each step of the procedure, thus leading to better clinical outcomes.

Silver-catalyzed three-component reaction of uracils, arylboronic acids, and selenium: synthesis of 5-arylselanyluracils.

Herein, we describe a simple and general multi-component synthesis of 5-arylselanyluracils by the regioselective C-H selenation of uracils. Reactions of uracils with arylboronic acid and Se powder in the presence of AgNO3 (10 mol%) at 120 °C under aerobic conditions afforded various 5-arylselanyluracils. The source of the introduced selanyl group was prepared from a commercially available arylboronic acid and Se powder in the reaction system, thereby ensuring a simple and efficient protocol. This reaction represents the first example of the synthesis of a 5-arylselanyluracil in a multi-component system.

Protective effect of Actinidia arguta in MPTP-induced Parkinson's disease model mice.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress-induced neuronal death has been identified as one of the major causes of nigrostriatal degeneration in PD. The fruit of Actinidia arguta (A. arguta), known as sarunashi in Japan, has been reported to show beneficial health effects such as antioxidant, anti-inflammatory, anti-mutagenic, and anticholinergic effects. In this study, we investigated the neuroprotective effects of A. arguta in 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP)-induced PD model mice. A. arguta juice was administered to 7-week-old C57BL/6J mice continuously for 10 days before the first MPTP injection. The degeneration of dopaminergic neurons in the substantia nigra was induced by MPTP (30 mg/kg, i. p.) once daily for five consecutive days. We found that the administration of A. arguta ameliorated MPTP-induced motor impairment and suppressed the MPTP-induced reductions of tyrosine hydroxylase-positive neurons and tyrosine hydroxylase protein expression in the substantia nigra. Our findings suggest that taking A. arguta could provide neuroprotection that delays or prevents the neurodegenerative process of PD.

Carotid artery stenting in a patient with an incidentally found double aortic arch: A case report.

Double aortic arch is a type of congenital vascular ring, which rarely presents in adults. Herein, we report a case of carotid artery stenting (CAS) accompanied by an incidentally found double aortic arch. A 77-year-old man presented with bilateral severe carotid artery stenosis. The patient underwent truncal 3D-computed tomography angiography (CTA) to evaluate the access route before CAS, which lead to the diagnosis of an asymptomatic double aortic arch. Referring to the 3D-CTA images, a guiding catheter was successfully navigated to the targeted common carotid artery via the transfemoral approach despite the challenging, complex anatomy of the aortic arch. CAS was performed in both carotid arteries under distal balloon protection, without any neurological deficits. Considering the possible presence of anatomical variants such as double aortic arch, preoperative evaluation of the access route would be efficient for prospective CAS patients.

Palladium-Catalyzed C-H Arylation of Benzofurans with Triarylantimony Difluorides for the Synthesis of 2-Arylbenzofurans.

Pd-catalyzed regioselective C-H arylation is a useful tool for the chemical modification of aromatic heterocycles and 2-arylbenzofuran derivatives are of interest as biologically active substances. Herein, the reaction of triarylantimony difluorides with benzofurans under aerobic conditions in 1,2-DCE, using 5 mol% Pd (OAc)2 and 2 eq. of CuCl2 at 80 °C, produced a variety of 2-arylbenzofurans in moderate-to-high yields. The reaction is sensitive to the electronic nature of the substituents on the benzene ring of the triarylantimony difluorides: an electron-donating group showed higher reactivity than an electron-withdrawing group. Single crystal X-ray analysis of tri(p-methylphenyl) antimony difluoride revealed that the central antimony atom exhibits trigonal bipyramidal geometry.

Proteomic identification of the proteins related to cigarette smoke-induced cardiac hypertrophy in spontaneously hypertensive rats.

Smoking increases the risk of cardiovascular diseases. The present study was designed to determine the effects of 2-month exposure to cigarette smoke (CS) on proteins in the left ventricles of spontaneously hypertensive rats (SHR) and to identify the molecular targets associated with the pathogenesis/progression of CS-induced cardiac hypertrophy. SHR and Wistar Kyoto rats (WKY) were exposed to CS at low (2 puffs/min for 40 min) or high dose (2 puffs/min for 120 min), 5 days a week for 2 months. Using the two-dimensional fluorescence difference gel electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry, we compared differences in the expression levels of proteins in the whole left ventricles induced by long-term smoking. High-dose CS mainly caused cardiac hypertrophy in SHR, but not WKY, but no change in blood pressure. Proteomic analysis identified 30 protein spots with significant alterations, with 14 up-regulated and 16 down-regulated proteins in the left ventricles of CS-exposed SHR, compared with control SHR. Among these proteins, two members of the heat shock proteins (HSP70 and HSP20) showed significant up-regulation in the left ventricles of CS high-dose SHR, and the results were confirmed by western blot analysis. Our findings suggested that HSPs play an important role in regulation of CS-induced cardiac hypertrophy.

Proteomic analysis of the monkey hippocampus for elucidating ischemic resistance.

It is well-known that the cornu Ammonis 1 (CA1) sector of hippocampus is vulnerable for the ischemic insult, whereas the dentate gyrus (DG) is resistant. Here, to elucidate its underlying mechanism, alternations of protein oxidation and expression of DG in the monkey hippocampus after ischemia-reperfusion by the proteomic analysis were studied by comparing CA1 data. Oxidative damage to proteins such as protein carbonylation interrupt the protein function. Carbonyl modification of molecular chaperone, heat shock 70 kDa protein 1 (Hsp70.1) was increased remarkably in CA1, but slightly in DG. In addition, expression levels of nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase sirtuin-2 (SIRT2) was significantly increased in DG after ischemia, but decreased in CA1. Accordingly, it is likely that SIRT2 upregulation and negligible changes of carbonylation of Hsp70.1 exert its neuroprotective effect in DG. On the contrary, carbonylation level of dihydropyrimidinase related protein 2 (DRP-2) and l-lactate dehydrogenase B chain (LDHB) were slightly increased in CA1 as shown previously, but remarkably increased in DG after ischemia. It is considered that DRP-2 and LDHB are specific targets of oxidative stress by ischemia insult and high carbonylation levels of DRP-2 may play an important role in modulating ischemic neuronal death.

Phenotypic analysis of human CYP2C9 polymorphisms using fluorine-substituted tolbutamide.

To investigate the effect of fluorine substitution on tolbutamide (TB) hydroxylation catalyzed by CYP2C9, the hydroxylation of TB and its fluorinated derivative 3'-fluoro-tolbutamide (3'-F-TB) by recombinant human CYP2C9*1, CYP2C9*2, and CYP2C9*3 was analyzed. In general, fluorine substitution near the metabolic site may decrease enzymatic oxidation owing to its electron-withdrawing nature. Fluorine substitution reduced the Michaelis-Menten-derived Km of 4'-hydroxylation of TB catalyzed by CYP2C9*1 from 115 (TB) to 77 (3'-F-TB) µM. In the case of TB hydroxylation catalyzed by CYP2C9*2, the Km value of TB was also reduced by fluorine substitution from 129 to 88 µM. The greatest effect of fluorine substitution on the Km in TB hydroxylation was observed in the catalysis by CYP2C9*3, in which the Km value decreased from 287 to 117 µM. When a mixture containing TB and 3'-F-TB was hydroxylated by CYP2C9, the hydroxylated metabolite ratio in CYP2C9*3 was significantly increased compared with that in CYP2C9*1 and CYP2C9*2 (p < 0.01, Tukey-Kramer test). These results suggest that obtaining the metabolite profiles of fluorine-substituted analogs of the key substrate molecule may be useful as a new tool for phenotyping polymorphic CYP isoforms.

Idarubicin, an Anthracycline, Induces Oxidative DNA Damage in the Presence of Copper (II).

BACKGROUND/AIM: The aim of the present study was to investigate whether idarubicin (IDR) induces oxidative DNA damage in the presence of copper (II). MATERIALS AND METHODS: DNA damage was evaluated by pBR322 plasmid DNA cleavage. The formation of oxidative stress markers [O2 •- and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] was analysed. RESULTS: IDR induced DNA damage and O2 •- and 8-OHdG generation in the presence of copper (II). CONCLUSION: IDR induced oxidative DNA damage in the presence of copper (II). Since it has been reported that the concentration of copper in the serum of cancer patients is higher than that in healthy groups, IDR-induced oxidative DNA damage in the presence of copper (II) may play an important role in anticancer therapeutic strategies.

Hepatocarcinogen 4-methylquinoline induced G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (Muta™Mouse).

We have previously reported that quinoline increased the mutation frequency of the cII gene in the liver of lambda/lacZ transgenic mice (Muta™Mouse), and G:C to C:G transversions were the molecular signature of quinoline-induced mutations. 4-Methylquinoline (4-MeQ) has the highest mutagenicity among quinoline and isomeric methylquinolines according to the Ames test using Salmonella typhimurium TA 100, in the presence of rat liver microsomal enzymes. In this report, we examined the effect of 4-MeQ on mutagenesis in the lambda cII gene in the liver of the Muta™Mouse, and we analyzed the sequences of the mutated genes. The mutation frequency of the liver cII gene was seven times higher in 4-MeQ-treated mice than in control mice. Sequence analysis revealed that 4-MeQ primarily induced G:C to C:G transversions (37 of 45). The specificities of 4-MeQ for target organ and mutation pattern were very consistent with those of quinoline. Thus, we showed that 4-MeQ was also genotoxic in the liver of the Muta™Mouse, and as with quinoline, the G:C to C:G transversion was the molecular signature of the 4-MeQ-induced mutations.

The impact of budesonide inhalation suspension for asthma hospitalization: In terms of length of stay, recovery time from symptoms, and hospitalization costs.

BACKGROUND: Hospitalization is a major cause of medical expenditure for asthma. Budesonide inhalation suspension (BIS) may assist in reducing asthma-related symptoms in severe asthma exacerbation. However, its effectiveness for hospitalized patients remains poorly known. The objective of this study is to determine associations of BIS with asthma hospitalization. METHODS: We retrospectively analyzed 98 patients who were admitted to our hospital due to severe asthma exacerbation (24 treated with BIS in combination with procaterol) from April 2014 to January 2019. Length of stay, recovery time from symptoms (wheezes), and hospitalization costs were compared between the 2 groups according to clinical factors including the use of BIS and sings of respiratory infections (i.e. C-reactive protein, the presence of phlegm, and the use of antibiotics). Multivariate logistic regression analysis was performed to determine factors contributing to hospitalization outcomes. RESULTS: The use of BIS was associated with shorter length of stay, faster recovery time from symptoms, and more reduced hospitalization costs (6.0 vs 8.5 days, 2.5 vs 5.0 days, and 258,260 vs 343,350 JPY). Signs of respiratory infection were also associated with hospitalization outcomes. On a multivariate regression analysis, the use of BIS was a determinant of shortened length of stay and reduced symptoms and medical costs for asthma hospitalization along with signs of respiratory infection. CONCLUSIONS: BIS may contribute to shorten length of hospital stay and to reduce symptoms and medical expenditure irrespective of the presence or absence of respiratory infection.

Small airway inflammation is associated with residual airway hyperresponsiveness in Th2-high asthma.

Background: Asthma is characterized by airway inflammation, variable airflow obstruction, and airway hyperresponsiveness (AHR). Generally, AHR takes longer to resolve than does airflow obstruction or clinical symptoms. AHR occasionally persists despite adequate asthma treatment.Objective: To evaluate factors which associates with residual AHR in patients with seemingly remitted airway inflammation.Methods: Patients who exhibited high fractional exhaled nitric oxide (FeNO) levels (>25 ppb) at the first visit (Visit 1) and normalized FeNO levels (<25 ppb) after adequate asthma treatment, including inhaled corticosteroid administration (Visit 2), were analyzed. Patients underwent a blood test, FeNO and small airway/alveolar nitric oxide concentration (CANO) measurements and a methacholine challenge test (continuous inhalation method) at both visits. Clinical indices were compared between patients with and without residual AHR.Results: Fifty patients were analyzed. All exhibited high FeNO levels at Visit 1 [mean, 54.0 ppb (95% confidence interval, 42.4-65.5)] and improvement of FeNO levels at Visit 2 [20.4 (19.2-21.6)] (p < 0.0001). Thirty-three patients (66%) had remission of AHR at Visit 2. No significant differences were observed between patients with and without residual AHR in terms of FeNO levels, lung function parameters and blood eosinophil counts at both visits. CANO level at Visit 2 was the only factor that significantly differed between patients with residual AHR [2.7 (1.9-3.6)] and those who achieved AHR remission [0.8 (0.5-1.0)] (p < 0.0001).Conclusion: Small airway inflammation, as assessed by CANO, was associated with residual AHR in patients with Th2-high asthma.

Intracellular accumulation-independent cytotoxicity of pentavalent organoantimony compounds in cultured vascular endothelial cells.

As the field of utilization of organic-inorganic hybrid molecules expands, the toxicology of these compounds is becoming more important. We have shown previously that there is a strong correlation between cytotoxicity and intracellular accumulation detected as metal content, which is modulated by the substituents, of organic-inorganic hybrid molecules. In this study, we investigated the cytotoxicity of pentavalent organoantimony compounds with three phenyl groups on cultured vascular endothelial cells. The results indicated that the cytotoxicity of pentavalent organoantimony compounds was not correlated with the hydrophobicity and intracellular accumulation of these compounds. Therefore, we suggest that hydrophobicity and intracellular accumulation are not necessarily predictive of cytotoxicity in organic-inorganic hybrid molecules.

[Improvement of the Non-aqueous Titration Method in the Japanese Pharmacopoeia for a Purpose of Advancement in Experimental Safety Using Alternative Solvents].

In this study, we attempted to improve the non-aqueous titration method using N,N-dimethylformamide (DMF) in the Japanese Pharmacopoeia seventeenth edition (JP XVII) for advancement in experimental safety. As an alternative solvent for DMF, we demonstrate titrations using dimethyl sulfoxide (DMSO), which has similar properties as and much higher safety than DMF. Five drugs (i.e., acetohexamide, glibenclamide, sulfamethoxazole, tranilast, and furosemide) listed in JP XVII use DMF as a solvent for titrations with sodium hydroxide standard solution. For these drugs, we examined whether DMF can be replaced with DMSO in quantitative analyses. As a result, a quantification similar to that of the Pharmacopoeia protocol is possible by simply replacing DMF with DMSO or using a mixed solvent of DMSO and water.

Synthesis of benzo[d]imidazo[2,1-b]benzoselenoazoles: Cs2CO3-mediated cyclization of 1-(2-bromoaryl)benzimidazoles with selenium.

The synthesis of benzimidazo[2,1-b]benzoselenoazoles is described. The novel ring-closure reaction of 1-(2-bromoaryl)benzimidazoles with Se powder is promoted by Cs2CO3 (2 equiv) in DMF at 150 °C. Moreover, the obtained tetracyclic heterocycles are all novel compounds. Single-crystal X-ray analysis of the parent benzimidazo[2,1-b]benzoselenoazole revealed that the tetracyclic ring is almost planar. Absorption spectroscopy data of the benzimidazo[2,1-b]benzoselenoazoles showed the λmax was dependent on the number of rings.