Search

Huntington’s disease (HD) is caused by a CAG repeat expansion in the huntingtin (HTT) gene. CRISPR-Cas9 nuclease causes double-strand breaks (DSBs) in the targeted DNA that induces toxicity, whereas CRISPR interference (CRISPRi) using dead Cas9 (dCas9) suppresses the target gene expression without DSBs. Delivery of dCas9-sgRNA targeting CAG repeat region does not damage the targeted DNA in HEK293T cells containing CAG repeats. When this study investigates whether CRISPRi can suppress mutant HTT (mHTT), CRISPRi results in reduced expression of mHTT with relative preservation of the wild-type HTT in human HD fibroblasts. Although both dCas9 and Cas9 treatments reduce mHTT by sgRNA targeting the CAG repeat region, CRISPRi delays behavioral deterioration and protects striatal neurons against cell death in HD mice. Collectively, CRISPRi can delay disease progression by suppressing mHtt, suggesting DNA DSB-free CRISPRi is a potential therapy for HD that can compensate for the shortcoming of CRISPR-Cas9 nuclease.

Polydeoxyribonucleotide (PDRN) is an agonist that selectively stimulates adenosine A2A receptor (ADORA2A), which suppresses inflammatory responses. Ischemia/reperfusion (I/R) injury plays a major role in the pathogenesis of ischemic stroke by inducing neuroinflammation. Therefore, this study aimed to investigate the therapeutic effects of PDRN in an in vitro I/R injury model. The in vitro model was established with differentiated Neuro-2a cells under oxygen and glucose deprivation condition. The cells were treated with PDRN for 24 h under reoxygenation condition. As the results of RNA-seq transcriptome analysis, CSF1, IL-6, PTPN6, RAC2, and STAT1 were identified of its relation to the effect of PDRN on inflammatory responses in the model. To further investigate therapeutic effects of PDRN, RT-qPCR, western blotting, LDH assay, and TUNEL assay were performed. PDRN significantly reversed the expression of genes and proteins related to inflammatory responses. The elevated ADORA2A expression by PDRN treatment downregulated JAK/STAT pathway in the model. Furthermore, PDRN inhibited neuronal cell death in the model. Consequently, our results suggested that PDRN alleviated inflammatory responses through inhibition of JAK/STAT pathway by mediating ADORA2A expression and inhibited neuronal cell death in the model. These results provide significant insights into potential therapeutic approaches involving PDRN treatment for I/R injury.

Objective : Cervical surgery in patients with cervical spondylotic myelopathy (CSM) and cerebral palsy (CP) is challenging owing to the complexities of the deformity. We assessed factors affecting postoperative complications and outcomes after CSM surgery in patients with CP.Methods : Thirty-five consecutive patients with CP and CSM who underwent cervical operations between January 2006 and January 2014 were matched to 35 non-cerebral palsy (NCP) control patients. Postoperative complications and radiologic outcomes were compared between the groups. In the CP group, the Japanese Orthopaedic Association score; Oswestry neck disability index; modified Barthel index; and values for the grip and pinch, Box and Block, and Jebsen-Taylor hand function tests were obtained preand postoperatively and compared between those with and without postoperative complications.Results : Sixteen patients (16/35%) in the CP group and seven (7/35%) in the NCP group (p=0.021) had postoperative complications. Adjacent segment degeneration (p=0.021), postoperative motor weakness (p=0.037), and revisions (p=0.003) were significantly more frequent in the CP group than in the NCP group; however, instrument-related complications were not significantly higher in the CP group (7/35 vs. 5/35, p=0.280). The number of preoperative fixed cervical deformities were significantly higher in CP with postoperative complications (5/16 vs. 1/19, p=0.037). In the CP group, clinical outcomes were almost similar between those with and without postoperative complications.Conclusion : The occurrence of complications during the follow-up period was high in patients with CP. However, postoperative complications did not significantly affect clinical outcomes.

The use of prime editing-a gene-editing technique that induces small genetic changes without the need for donor DNA and without causing double strand breaks-to correct pathogenic mutations and phenotypes needs to be tested in animal models of human genetic diseases. Here we report the use of prime editors 2 and 3, delivered by hydrodynamic injection, in mice with the genetic liver disease hereditary tyrosinemia, and of prime editor 2, delivered by an adeno-associated virus vector, in mice with the genetic eye disease Leber congenital amaurosis. For each pathogenic mutation, we identified an optimal prime-editing guide RNA by using cells transduced with lentiviral libraries of guide-RNA-encoding sequences paired with the corresponding target sequences. The prime editors precisely corrected the disease-causing mutations and led to the amelioration of the disease phenotypes in the mice, without detectable off-target edits. Prime editing should be tested further in more animal models of genetic diseases.

Mutations in the X-linked methyl-CpG-binding protein 2 ( MECP2) gene were first described as a cause of Rett syndrome. MECP2 duplication can cause intellectual disability, developmental delay, severe feeding difficulties, and recurrent infections. Here, we report a Korean family with MECP2 duplication syndrome, which was previously misdiagnosed as cerebral palsy. A man in his early 30 s visited our clinic with intellectual disability, speech impairment, epilepsy, and progressive spasticity. He had been previously misdiagnosed with cerebral palsy, and had received orthopedic surgeries such as musculotendinous lengthening and derotational osteotomy. After the surgeries, he received comprehensive rehabilitation. Upon carefully checking his family history, we noted that his younger brother had similar symptoms. Next-generation sequencing revealed whole exon duplication in MECP2 in both the patient and his brother; their mother also had this genetic mutation but was asymptomatic. Early diagnosis is essential for improving the success of MECP2 duplication syndrome treatment. Individuals with MECP2 duplication syndrome should be referred to specialists to manage multidisciplinary symptoms and to regularly check for complications that are common in this syndrome.

Two of the primary features of Parkinson's disease (PD) are the accumulation of α-synuclein (α-Syn) and the depletion of lysosomal-associated membrane protein 1 (LAMP1) in the brain. Beneficial effects of environmental enrichment (EE) have been reported on the activation of lysosomal function and the amelioration of PD symptoms. Furthermore, Reelin could be a novel therapeutic target in PD. Hence, in this study, we validated the effects of EE on the activation of LAMP1 via Reelin in PD. Heterogeneous α-Syn (A53T)-overexpressing transgenic mice (age 6 and 16 months) were exposed to EE for 8 weeks. After motor and cognitive tests, brain tissues were obtained from mice and subjected to immunohistochemistry and molecular analyses. EE ameliorated motor and non-motor symptoms, protected dopamine neurons, and reduced pathological α-Syn accumulation in the early stage of PD. Striatal Reelin levels were altered depending on the disease stage and regulated by EE in PD mice. To elucidate the underlying mechanism of the effect of EE on PD, we performed further molecular and cellular analyses using activated preformed fibril (PFF)-induced SH-SY5Y cells, an in vitro model of PD, which were treated with recombinant Reelin protein and a Reelin blocker, CR-50. The CR-50 increased pathological α-Syn accumulation and accelerated dopamine neuronal degeneration by decreasing LAMP1 in the PFF-induced PD model. Our results showed that Reelin increased LAMP1 after EE and decreased pathological α-Syn accumulation, thus protecting dopamine neurons from degeneration in the striatum and substantia nigra, and ameliorating neurobehavioral deficits. These results suggest that Reelin is a promising target in treating histopathological changes and improving behavioral symptoms associated with PD.

Environmental enrichment (EE) is a promising therapeutic strategy in improving metabolic and neuronal responses, especially due to its non-invasive nature. However, the exact mechanism underlying the sex-differential effects remains unclear. The aim of the current study was to investigate the effects of EE on metabolism, body composition, and behavioral phenotype based on sex. Long-term exposure to EE for 8 weeks induced metabolic changes and fat reduction. In response to the change in metabolism, the level of βHB were influenced by sex and EE possibly in accordance to the phases of estrogen cycle. The expression of β-hydroxybutyrate (βHB)-related genes and proteins such as monocarboxylate transporters, histone deacetylases (HDAC), and brain-derived neurotrophic factor (BDNF) were significantly regulated. In cerebral cortex and hippocampus, EE resulted in a significant increase in the level of βHB and a significant reduction in HDAC, consequently enhancing BDNF expression. Moreover, EE exerted significant effects on motor and cognitive behaviors, indicating a significant functional improvement in female mice under the condition that asserts the influence of estrogen cycle. Using an ovariectomized mice model, the effects of EE and estrogen treatment proved the hypothesis that EE upregulates β-hydroxybutyrate and BDNF underlying functional improvement in female mice. The above findings demonstrate that long-term exposure to EE can possibly alter metabolism by increasing the level of βHB, regulate the expression of βHB-related proteins, and improve behavioral function as reflected by motor and cognitive presentation following the changes in estrogen level. This finding may lead to a marked improvement in metabolism and neuroplasticity by EE and estrogen level.

Early rehabilitation presents favorable outcomes for stroke patients. However, the optimal strategy for early rehabilitation is unclear currently due to the current limitation in the data on the effects of early initiation of rehabilitation based on voluntary exercise. During the hyperacute phase of ischemic stroke, upregulation of Fas/FasL-mediated apoptosis is observed. Environmental enrichment (EE) is a therapeutic paradigm for laboratory animals that consists of complex combinations of physical, cognitive, social stimuli and voluntary exercise. Few studies delineated the effect of EE on apoptosis in an experimental model of hyperacute stroke. The aim of the study is to determine whether hyperacute exposure to EE can effectively regulate Fas/FasL-mediated apoptosis following hypoxic-ischemic brain injury and improve neurobehavioral function. C57Bl/6 mice were randomly assigned to either EE or standard cage (SC) within 3 hours or on day 3 after hypoxic-ischemic brain injury for 2 weeks. Neurobehavioral tests, transcriptome analysis, Western Blot, and immunohistochemistry were performed in the brain samples of cerebral cortex and hippocampus, and total infarct volume was calculated. Compared with SC, hyperacute exposure to EE was associated with greater improvement in anxiety, motor function, cognitive ability, reduced total infarct volume, and decreased neuronal death. It significantly downregulated Fas/FasL-mediated apoptosis, decreased expression of Fas, FADD, cleaved caspase-8/caspase-8, cleaved caspase-3/caspase-3, as well as Bax/Bcl-2 in both regions. Overall, the results of this study demonstrates that the early exposure to EE is a neuroprotective therapeutic translation for stroke rehabilitation through effective inhibition of extrinsic as well as intrinsic apoptotic pathways.

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

Several Streptococcus pyogenes Cas9 (SpCas9) variants have been developed to improve an enzyme’s specificity or to alter or broaden its protospacer-adjacent motif (PAM) compatibility, but selecting the optimal variant for a given target sequence and application remains difficult. To build computational models to predict the sequence-specific activity of 13 SpCas9 variants, we first assessed their cleavage efficiency at 26,891 target sequences. We found that, of the 256 possible four-nucleotide NNNN sequences, 156 can be used as a PAM by at least one of the SpCas9 variants. For the high-fidelity variants, overall activity could be ranked as SpCas9 ≥ Sniper-Cas9 > eSpCas9(1.1) > SpCas9-HF1 > HypaCas9 ≈ xCas9 >> evoCas9, whereas their overall specificities could be ranked as evoCas9 >> HypaCas9 ≥ SpCas9-HF1 ≈ eSpCas9(1.1) > xCas9 > Sniper-Cas9 > SpCas9. Using these data, we developed 16 deep-learning-based computational models that accurately predict the activity of these variants at any target sequence. Deep-learning models predict the Cas9 variant with optimal activity and specificity for any target sequence.

This study aimed to investigate the differences in gait outcomes of young adults with cerebral palsy (CP) following rhythmic auditory stimulation (RAS) with different types of cueing. A total of 13 ambulatory adults with CP were recruited. The participants were assigned to receive either RAS with simple chords or RAS with complex chords. Each participant received 30-min individual sessions three times per week for 4 weeks. In the simple RAS group, basic chords were used for cueing. In the complex RAS group, the diversified chords were adopted from patients' preferred music. At pre- and posttest, spatiotemporal and kinematic parameters and the range of motion (ROM) for each joint during a gait cycle were collected. After RAS, cadence, velocity, and stride length significantly increased, but no significant group effect was found. Meanwhile, regarding kinematic parameters, a significant interaction effect between time and group was observed with the angle of plantar flexion in the preswing phase and ROM in the ankle. The complex RAS group showed increased maximal ankle plantar flexion in the preswing phase. These results demonstrated that the primary agent for gait control is rhythm, while perception of music facilitates patient engagement in walking differently depending on the level of musical elements.

Lateral cephalometry has been widely used for skeletal classification in orthodontic diagnosis and treatment planning. However, this conventional system, requiring manual tracing of individual landmarks, contains possible errors of inter- and intravariability and is highly time-consuming. This study aims to provide an accurate and robust skeletal diagnostic system by incorporating a convolutional neural network (CNN) into a 1-step, end-to-end diagnostic system with lateral cephalograms. A multimodal CNN model was constructed on the basis of 5,890 lateral cephalograms and demographic data as an input. The model was optimized with transfer learning and data augmentation techniques. Diagnostic performance was evaluated with statistical analysis. The proposed system exhibited >90% sensitivity, specificity, and accuracy for vertical and sagittal skeletal diagnosis. Clinical performance of the vertical classification showed the highest accuracy at 96.40 (95% CI, 93.06 to 98.39; model III). The receiver operating characteristic curve and the area under the curve both demonstrated the excellent performance of the system, with a mean area under the curve >95%. The heat maps of cephalograms were also provided for deeper understanding of the quality of the learned model by visually representing the region of the cephalogram that is most informative in distinguishing skeletal classes. In addition, we present broad applicability of this system through subtasks. The proposed CNN-incorporated system showed potential for skeletal orthodontic diagnosis without the need for intermediary steps requiring complicated diagnostic procedures.

Human norovirus (HNoV) GII.4 and Vibrioparahaemolyticus may be found in sea squirts. Antimicrobial effects of floating electrode-dielectric barrier discharge (FE-DBD) plasma (5–75 min, N2 1.5 m/s, 1.1 kV, 43 kHz) treatment were examined. HNoV GII.4 decreased by 0.11–1.29 log copy/μL with increasing duration of treatment time, and further by 0.34 log copy/μL when propidium monoazide (PMA) treatment was added to distinguish infectious viruses. The decimal reduction time (D1) of non-PMA and PMA-treated HNoV GII.4 by first-order kinetics were 61.7 (R2 = 0.97) and 58.8 (R2 = 0.92) min, respectively. V.parahaemolyticus decreased by 0.16–1.5 log CFU/g as treatment duration increased. The D1 for V. parahaemolyticus by first-order kinetics was 65.36 (R2 = 0.90) min. Volatile basic nitrogen showed no significant difference from the control until 15 min of FE-DBD plasma treatment, increasing after 30 min. The pH did not differ significantly from the control by 45–60 min, and Hunter color in “L” (lightness), “a” (redness), and “b” (yellowness) values reduced significantly as treatment duration increased. Textures appeared to be individual differences but were not changed by treatment. Therefore, this study suggests that FE-DBD plasma has the potential to serve as a new antimicrobial to foster safer consumption of raw sea squirts.

This study aimed to objectively assess YouTube videos’ quality, reliability, and information delivery capability regarding novel spinal muscular atrophy treatments. Using the keywords “nusinersen”, “spinraza”, “ridisplam”, “evrysdi”, “onasemnogene abeparvovec”, and “zolgensma”, we were able to retrieve and screen 360 videos before settling on a final sample of 99 on 25 September 2022. Then, two independent raters used the mDISCERN and GQS instruments to evaluate the videos’ reliability and quality and the Information Delivery Capability (IDC) score to assess the videos’ accuracy and patient-friendliness. The quality, reliability, and information delivery capability of the videos about the new treatment for SMA were quite heterogeneous, with an average mDISCERN, GQS, and IDC score of 3.172 ± 0.899, 2.980 ± 1.025, and 4.141 ± 1.747, respectively. In-depth analysis showed that healthcare expert videos that explained contents while showing infographic supplements had good quality, reliability, and information delivery capability. As YouTube is already a dominant media platform, the public may obtain new information about novel therapeutics for SMA through YouTube. It is necessary to consider how SMA patients and caregivers can choose trusted sources with reliable information on YouTube, and our results can provide clues. Additionally, experts should strive to provide more accurate, reliable, and patient-oriented videos.

Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency, While prime editing is a promising technology, PE2 systems often have low efficiency. Here the authors fuse a Rad51 DNA-binding domain to create hyPE2 with improved editing efficiency.

Repetitive magnetic stimulation (rMS) has been suggested as a non-invasive treatment for various neurological or psychiatric diseases. Contrary to the application previously used, the purpose of the present study was to elucidate whether low-frequency rMS could suppress tumor progression in in vitro and in vivo neuroblastoma models, and to explore the underlying mechanisms. The results demonstrated that low-frequency rMS treatment significantly suppressed cell proliferation and tumor progression in the models. Moreover, low-frequency rMS treatment downregulated the Wnt/β-catenin signaling pathway and induced apoptosis. The Wnt/β-catenin signaling pathway activator, Wnt agonist, was found to counteract the effect of low-frequency rMS treatment, while the Wnt/β-catenin signaling pathway inhibitor, Wnt antagonist, exhibited a tumor suppression effect, similar to the effect of low-frequency rMS treatment. Taken together, our data demonstrated that low-frequency rMS treatment suppressed neuroblastoma progression by downregulating the Wnt/β-catenin signaling pathway, suggesting that low-frequency rMS treatment may be a potential therapeutic strategy for the tumor suppression.

Summary: Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients. : Hemophilia A (HA) is caused by various genetic mutations within the blood coagulation factor VIII (FVIII) gene. In this article, Kim and colleagues attempt the targeted insertion of the FVIII gene into the human H11 site in two types of HA patient iPSCs. This approach may offer a universal therapeutic method for correcting all genetic variants found in HA patients. Keywords: genome editing, H11 locus, CRISPR/Cas9, hemophilia, iPSCs

Cell therapeutic agents for treating degenerative brain diseases using neural stem cells are actively being developed. However, few systems have been developed to monitor in real time whether the transplanted neural stem cells are actually differentiated into neurons. Therefore, it is necessary to develop a technology capable of specifically monitoring neuronal differentiation in vivo. In this study, we established a system that expresses cell membrane-targeting red fluorescent protein under control of the Synapsin promoter in order to specifically monitor differentiation from neural stem cells into neurons. In order to overcome the weak expression level of the tissue-specific promoter system, the partial 5′ UTR sequence of Creb was added for efficient expression of the cell surface-specific antigen. This system was able to track functional neuronal differentiation of neural stem cells transplanted in vivo, which will help improve stem cell therapies.

IntroductionMegaShield®is a newly developed temperature-sensitive anti-adhesive containing micronized acellular dermal matrix. The aim of this study was to investigate the efficacy and safety of MegaShield®compared with Guardix-SG®in the prevention of adhesions in patients undergoing bilateral total thyroidectomy.MethodWe conducted a multicenter trial between October 2018 and March 2020 in patients undergoing total thyroidectomy. The patients were randomly assigned to either the MegaShield®group or the Guardix-SG®group. The primary outcome was the esophageal movement using marshmallow six weeks after the surgery and the secondary outcome was the assessed adhesion score. The safety assessment was also evaluated.ResultsThe study included 70 patients each in the MegaShield®and control (Guardix-SG®) groups. Baseline clinical characteristics, the mean score of marshmallow esophagography, and the sum of adhesion scores were not statistically different between the two groups. Inferiority test demonstrated that the efficacy of MegaShield®is not inferior to that of Guardix-SG®. There were no device-related complications in both groups.ConclusionThe efficacy and safety of MegaShield®were not inferior than those of Guardix-SG®. MegaShield®demonstrated the potential of ADM as a potential future anti-adhesive agent.Trial registrationThe name of trial registry CRIS (Clinical Research Information Service)https://cris.nih.go.kr/cris/index.jsp. (The full trial protocol can be accessed) Registration number:KCT0003204.

Prime editing can induce any small-sized genetic change without donor DNA or double strand breaks. However, it has not been investigated whether prime editing is possible in postnatal animals. Here we delivered prime editors 2 and 3 into a mouse model of hereditary tyrosinemia, a genetic liver disease, using hydrodynamic injection, which corrected the disease-causing mutation and rescued the phenotype. We also achieved prime editing in the retina and retina pigment epithelium in wild-type mice by delivering prime editor 3 using trans-splicing adeno-associated virus. Deep sequencing showed that unintended edits at or near the target site or off-target effects were not detectable except for low levels (0% to 1.2%) of indels when PE3, but not PE2, was used. Our study suggests that precise, prime editor-mediated genome editing is possible in somatic cells of adult animals.

Parkinson’s disease (PD) is a prevalent motor disease caused by the accumulation of mutated α-synuclein (α-Syn), however, its early stages are also characterized by non-motor symptoms, such as olfactory loss, cognitive decline, depression, and anxiety. The therapeutic effects of environmental enrichment (EE) on motor recovery have been reported, but its effects on non-motor symptoms remain unclear. Herein, we reveal the beneficial effects of EE on PD-related non-motor symptoms and changes in synaptic plasticity in the nucleus accumbens. To investigate its therapeutic effects in the early phase of PD, we randomly assigned eight-month-old mice overexpressing human A53T (hA53T) α-Syn to either the EE or standard condition groups for two months. Next, we performed behavioral tests and biochemical and histological analyses at 10 months of age. EE significantly alleviated locomotor hyperactivity and anxiety during the early stages of PD. It normalized the levels of tyrosine hydroxylase, phosphorylated and oligomeric α-Syn, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex-forming proteins, including synaptosomal-associated protein, 25 kDa, syntaxin1, and vesicle-associated membrane protein 2 (VAMP2). Moreover, the interactions between VAMP2 and pSer129 α-Syn were markedly reduced following EE. The restoration of synaptic vesicle transportation status may underlie the neuroprotective effects of EE in hA53T α-Syn mice.

BackgroundAlthough Parkinson’s disease (PD) is one of the prevalent motor diseases caused by the accumulation of mutated α-Synuclein (α-Syn), it is characterized by non-motor symptoms such as olfactory loss, cognitive decline, depression and anxiety in the early stage of the disease. Environmental enrichment (EE) provides a complex environment comprising physical, cognitive, and social stimuli that improve synaptic plasticity and behavioral functions. The therapeutic effects of EE on behavioral recovery of motor function in a mouse model of PD pathology have been reported. However, the effects of EE on non-motor symptoms in human A53T α-Syn overexpressing transgenic (hA53T α-Syn) mice have yet to be determined. In this study, we revealed the beneficial effect of EE on changes in synaptic plasticity in the striatum and nucleus accumbens (NAc) during the initial phase of PD.MethodTo investigate therapeutic effects of EE in abnormalities during the early phase of PD, we randomly assigned eight-month-old hA53T α-Syn mice to either EE (PD-EE) or standard conditions (PD-SC) for two months. Next, we performed behavioral tests, biochemical and histological analysis at 10 months of age.ResultsEE significantly alleviated locomotor hyperactivity and anxiety in the early stage of PD. EE normalized the level of tyrosine hydroxylase (TH), phosphorylated and oligomeric forms of α-Syn (pSer129 α-Syn), and SNARE complex-forming proteins including SNAP-25, Syntaxin1 and VAMP2. Moreover, the close link between VAMP2 and pSer129 α-Syn was significantly reduced after exposure to EE.ConclusionsOur results showed that EE attenuates aversive mood state on the early stage of PD mouse model. These results were parallel with the reduced expression of pathological α-Syn and the increased expression of neurotransmitter transporter proteins in EE. Interestingly, the interaction between pSer129 α-Syn and VAMP2 also decreased in EE. The restoration of synaptic vesicle transportation status may be responsible for the neuroprotective effects of EE in hA53T α-Syn mice.

Factor VII (FVII) deficiency is the most common among the rare bleeding disorders, which is caused by mutations in coagulation factor VII. Clinical features caused by FVII deficiency vary from mild or asymptomatic to fatal cerebral hemorrhage. We generated an induced pluripotent stem cell (iPSC) line, YCMi002-A, from FVII deficiency patient-derived fibroblasts. YCMi002-A cells are characterized by novel compound heterozygous mutations. The c.345C > A; p.C115X is well known and the second one, c.1276C > T; p.Q426X, remains novel. YCMi002-A cells may help researchers to understand correlation between these mutations and the symptoms of FVII deficiency.

DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."

The overproduction and purification of human proteins is a requisite of both basic and medical research. Although many recombinant human proteins have been purified, current protein production methods have several limitations; recombinant proteins are frequently truncated, fail to fold properly, and/or lack appropriate post-translational modifications. In addition, such methods require subcloning of the target gene into relevant plasmids, which can be difficult for long proteins with repeated domains. Here we devised a novel method for target protein production by introduction of a strong promoter for overexpression and an epitope tag for purification in front of the endogenous human gene, in a sense performing molecular cloning directly in the human genome, which does not require cloning of the target gene. As a proof of concept, we successfully purified intact human Reelin protein, which is lengthy (3460 amino acids) and contains repeating domains, and confirmed that it was biologically functional.

Background: White matter atrophy has been shown to precede the massive loss of striatal GABAergic neurons in Huntington’s disease (HD). The HD-induced white matter atrophy is associated with motor deficits. In vivo reprogramming toward a plastic state has emerged as a new approach for treating neurological diseases. Particularly, octamer-binding transcription factor 4 (OCT4) can induce myelin repair and functional recovery. This study investigated the effects of in situ expression of reprogramming factor OCT4 on behavioral performances, neural stem cell (NSC) niche activation in the subventricular zone (SVZ) and induction of cell fate specific to the changed microenvironment of HD. Methods: R6/2 mice, a transgenic mouse model of HD, randomly received adeno-associated virus serotype 9 (AAV9)-OCT4, AAV9-Null, or phosphate-buffered saline in both lateral ventricles at 4 weeks of age. To evaluate the behavioral improvement, rotarod test and grip strength test were performed at regular intervals. To investigate the expression of oligodendrocyte progenitor cell (OPC)-related genes, real-time quantitative reverse transcription PCR (qRT-PCR) and immunohistochemistry were performed. Next, we assessed the amelioration of myelination deficits via transmission electron microscope (TEM) and magnetic resonance imaging (MRI) at 13 weeks of age. Finally, we confimed striatal neuroprotecion by qRT-PCR and confocal microscopy.Results: The AAV9-OCT4 group displayed significantly improved rotarod performance and grip strength compared to the control groups. Following AAV9-OCT4 treatment, the number of newly generated NSCs and OPCs was significantly increased in the SVZ, and the expression of OPC-related genes such as NG2, Olig2, PDGFRα, Wnt3 and myelin regulatory factor (MYRF), and glial cell-derived neuroprotective factor (GDNF) was significantly increased. Further, the amelioration of myelination deficits in the corpus callosum was observed through TEM and MRI, and striatal DARPP32+ GABAergic neurons significantly increased in the AAV9-OCT4 group.

Difficulties with speech and swallowing occur in patients with Parkinsonism. Lee Silverman Voice Treatment (LSVT) is proven as an effective treatment for speech and swallowing function in idiopathic Parkinson’s disease (IPD). The effect of LSVT on swallowing function in multiple system atrophy-cerebellar type (MSA-C) is unknown. We sought to determine LSVT’s effect on swallowing function in MSA-C patients compared to IPD patients. LSVT-LOUD was performed on 13 patients with Parkinsonism (6 IPD and 7 MSA-C). Maximum phonation time (MPT), voice intensity, Speech Handicap Index-15 (SHI-15), Swallowing-Quality of Life (SWAL-QOL), National Institutes of Health-swallowing safety scale (NIH-SSS), and videofluoroscopic dysphagia scale (VDS) before and after LSVT were analyzed and reevaluated three months after treatment. The IPD and MSA-C groups showed significant improvements in overall speech and swallowing measures after LSVT. In particular, pharyngeal phase score and total score of VDS improved significantly in both groups. A two-way repeated-measure ANOVA revealed a significant main effect for time in the MPT, voice intensity, NIH-SSS, pharyngeal phase score and total score of VDS, psychosocial subdomain of SHI-15, and SWAL-QOL. The MSA-C group experienced less overall improvement in swallowing function, but the two groups had an analogous pattern of improvement. In conclusion, LSVT is effective for enhancing swallowing function, particularly in the pharyngeal phase, in both IPD and MSA-C patients. This study demonstrated that LSVT elicits significant improvements in MSA-C patients. We deemed LSVT to be an effective treatment for IPD and MSA-C patients who suffer from dysphagia.

Background Individuals with cerebral palsy (CP) experience bone loss due to impaired weight bearing. Despite serious complications, there is no standard medication. Objective To develop a new pharmacological agent, we performed a series of studies. The primary aim was to develop an animal model of CP to use our target medication based on transcriptome analysis of individuals with CP. The secondary aim was to show the therapeutic capability of collagen-binding peptide (CBP) in reversing bone loss in the CP mouse model. Methods A total of 119 people with CP and 13 healthy adults participated in the study and 140 mice were used for the behavioral analysis and discovery of therapeutic effects in the preclinical study. The mouse model of CP was induced by hypoxic-ischemic brain injury. Inclusion and exclusion criteria were established for CBP medication in the CP mouse model with bone loss. Results On the basis of clinical outcomes showing insufficient mechanical loading from non-ambulatory function and that underweight mainly affects bone loss in adults with CP, we developed a mouse model of CP with bone loss. Injury severity and body weight mainly affected bone loss in the CP mouse model. Transcriptome analysis showed SPP1 expression downregulated in adults with CP who showed lower bone density than healthy controls. Therefore, a synthesized CBP was administered to the mouse model. Trabecular thickness, total collagen and bone turnover activity increased with CBP treatment as compared with the saline control. Immunohistochemistry showed increased immunoreactivity of runt-related transcription factor 2 and osteocalcin, so the CBP participated in osteoblast differentiation. Conclusions This study can provide a scientific basis for a promising translational approach for developing new anabolic CBP medication to treat bone loss in individuals with CP.

Therapeutic goals for hemiplegic patients with neurological impairments are mainly focused on improving their independent lives. Based on the previously reported effectiveness of Wii Fit balance training, this study investigated the most influential outcomes after long-term intensive training (including balance and functional factors) on quality of life in hemiplegic patients. The intervention group (n = 21) received Nintendo Wii Fit balance training under supervision, and control group (n = 20) received conventional balance training by an occupational therapist. Two groups were matched based on age and onset duration. Both groups received a total of 15 treatments for 30 min per session, twice a week for 8 weeks. There were significant improvements not only in balance confidence and activities of daily living, but also in body composition, such as fat proportion and metabolic rate, in the intervention group compared to the control group (p < 0.05). In particular, balance confidence significantly affected EuroQoL Visual Analogue Scale according to stepwise multiple regression analyses in this study. These results demonstrated that Wii Fit balance training using virtual reality improved the quality of life of hemiplegic patients while overcoming the asymmetrical weight distribution of the affected side via the self-modulating biofeedback exercises.

This study compared the quality and drying characteristics of dried king oyster mushroom (Pleurotus eryngii), shiitake mushroom (Lentinus edodes GNA01) and maitake mushroom (Grifola frondosa) slices obtained by sundrying, hot-air drying (40, 50, 60℃), low-temperature vacuum drying (20, 25, 30℃), heat-pump dehumidifying drying (30, 40, 50℃), and freeze drying. The quality changes investigted included color, browning degree, hardness, general components, β-glucan. Color changes in king oyster mushroom during freeze drying were less than that between control (raw) and treated mushrooms. Compared with other drying methods, low-temperature vacuum drying of shiitake mushroom resulted in less color changes. Browning degree differed significantly between the different drying methods. As temperature increased, low-temperature vacuum drying resulted in decreased browning while heat-pump dehumidifying drying resulted in increased browning. In king oyster mushroom, hardness increased with temperature under hot-air drying, low-temperature vacuum drying and heat-pump dehumidifying drying. In shiitake mushroom, hardness increased with increasing temperature under hot-air drying and heat-pump dehumidifying drying. β-glucan content after drying was found to be between 28.29 and 39.39% in king oyster mushroom, 23.05 and 29.48% in shiitake mushroom and 16.10 and 24.51% in maitake mushroom.

배경 및 목적: 말장애지수(Speech Handicap Index, SHI)는 말 평가에 필수적으로 실시되는 자기보고 평가로서, 구강이나 구인두암 환자의 말 기능과 심리사회적 기능을 평가하는 데 특화되어 개발된 도구이다. SHI가 말 문제를 평가하기에 적합하게 개발되었음에도 불구하고 30개라는 전체 문항 수는 시간적인 제약을 받는다. 따라서 이 연구에서는...

배경 및 목적: 대부분의 파킨슨병 환자들은 본인들의 말 문제를 스스로 인식하므로, 그로 인해 좌절감, 무능감을 느끼고 삶의 질이 저하된다. 국내외적으로 파킨슨병 환자의 일반적인 삶의 질에 관한 다양한 연구가 이루어지고 있지만 파킨슨병 환자의 말 문제에 특화된 삶의 질 연구는 그리 많지 않다. 방법: 본 연구는 파킨슨병 환자 48명을 대상으로...

Purpose Pain Scrambler therapy is a patient-specific electrocutaneous nerve stimulation device. Burn pruritus is a common form of chronic and disabling neuropathic pain that is often difficult to treat effectively. Pruritus is mediated by histamines, which are effector molecules stored in mast cells and released locally during injury or inflammation. Burn pruritus may be accompanied by peripheral neuropathic pain, which may result from injury to sensory nerves that hampers conductance of neuronal messages along the large A and small C afferent fibers to the spinal cord. In this study, we investigated the effect of pain Scrambler therapy on burn scar pruritus. Methods Sixteen subjects were recruited to participate in this study. The subjects complained of severe pruritus that was rated at least 5 on the visual analogue scale (VAS), despite treatments with antihistamines, gabapentin medication, and other physical modalities. Each Scrambler Therapy with the MC-5A Pain Scrambler Therapy® technology device was performed for 40 min daily (Monday through Friday) for 10 consecutive days. The stimulus was increased to the maximum intensity bearable by the individual patient without causing any additional pain or discomfort. The numerical rating scale (NRS), 5-D Itch Scale, and Leuven Itch Scale were administered and evaluated immediately before Scrambler therapy, and then immediately after 5 and 10 therapy sessions. Results For all 16 patients, NRS showed mean values of 6.75 ± 1.13 before therapy, 5.06 ± 1.53 after 5 sessions, and 4.13 ± 1.45 after 10 sessions. The NRS values before therapy and after 10 sessions were significantly different (p

배경 및 목적: 독립적인 생활이 여전히 가능한 치매 환자조차도 인지 기능의 저하로 인해 숫자 처리와 계산에서 어려움을 보일 수 있다. 본 연구에서는 치매 환자군이 정상군에 비해 숫자 처리와 계산 수행 시 오류율과 오류 유형에서 어떤 차이를 보이는지 살펴보았다. 방법: 알츠하이머성 치매 환자 및 일반 노인 각 16명씩을 대상으로 숫자 표기법(기수법,...

배경 및 목적: 알츠하이머성 치매(Alzheimer s Disease, AD)로 인한 한글 읽기장애가 흔히 관찰됨에도 불구하고 그에 대한 연구는 미비한 실정이다. 본 연구에서는 읽기 경로 특성과 관련하여 알츠하이머성 치매군의 단어 어휘성 및 규칙성에 따른 실독증 특성을 밝히고자 하였다. 방법: 전반적 퇴화척도 GDS 3-6에 해당하는 치매군 20명과 정상군...

배경 및 목적: 본 연구에서는 점진적 소자증을 나타내는 특발성 파킨슨병 환자와 정상 대조군을 대상으로 구 쓰기와 한 음절 반복 쓰기를 통하여 가로쓰기와 세로쓰기 간의 글자 크기 변화율을 비교하였다. 방법: 본 연구 대상은 H&Y 단계가 2 이상인 특발성 파킨슨병 환자 중 점진적 소자증을 나타내는 11명과 이들의 연령과 성별, 학력, 손잡이 등을...