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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.

The genus Hapalochlaena, including the blue-lined octopus Hapalochlaena fasciata (H. fasciata), is highly toxic. Venomous, blue-lined octopuses were recently found in Korea, but their toxicity, toxin composition, and distribution remain largely unknown. Here we estimated the geographic distribution of the organisms along the Korean coast and clarified their toxicity. Tetrodotoxin (TTX) was present in all three specimens of H. fasciata examined, although the toxicity varied largely between individuals. The mean TTX concentration in the whole body of the three specimens was 6.5 ± 2.2 μg/g (range 3.3–8.5 μg/g). Among the body parts examined, the salivary glands exhibited the highest concentration (22.4 ± 9.7 μg/g). From 2012 to 2021, 26 individuals were obtained nearly every month from different regions of the Korean coast. A non-fatal case of a blue-lined octopus bite was reported along the Korean coast in June 2015. This is the first report on the widespread distribution of blue-lined octopuses on the Korean coast and TTX detection. The widespread distribution of the TTX-bearing H. fasciata along the Korean coast within the temperate zone indicates that the species may soon become a serious health issue in Korea. The toxicity of this species is also a potentially significant human health risk.

This study was performed to review which mHealth apps that improve brace-wearing compliance are currently available, and to carry out their quality assessments by listing their functionalities. We found ten mHealth apps in the literature review and commercial mHealth apps market (Google Play and App store). Then, the quality of these apps was evaluated by their transparency, health content, excellent technical content, security/privacy, issues of usability, and subjective ratings (THESIS) scale, and the functionalities of the included apps were reviewed. Regarding these functionalities, four categories (data acquisition, compliance enhancement, educational components, and additional functionalities) and twelve subcategories were identified. The mean overall quality of the apps was 3.00 out of 5. Although four of the apps achieved a score of 3.0 or more for their overall quality, indicating an acceptable quality, none of the apps scored higher than 4.0, which indicated a high or excellent quality. According to the sections, the transparency section had the highest rating (3.92) and the security/privacy section received the lowest rating (2.02). Given that the overall quality of current mHealth apps was not high, and their potential to motivate patients with idiopathic scoliosis to adhere to their bracing treatment, it is necessary to develop high-quality apps with appropriate functionalities for supporting brace treatment.

The safety of seafood is a critical public health concern in Korea because of the high rate of raw seafood consumption. We investigated the prevalence and antimicrobial resistance of fecal-associated bacteria (Escherichia coli, Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus) in 50 seawater samples and 48 aquatic animals collected from aquaculture farms along the Korean coast in 2018. Of them, E. coli was the most prevalent in seawater (24.0%) and aquatic animals (18.8%). Although more than 80.0% of E. coli isolates were sensitive to 9 of 15 antimicrobials, approximately 20.0% of the isolates were resistant to 4 antimicrobials, including tetracycline and streptomycin. Enterococcus spp. isolates (2.7–32.0%) were resistant to only 5 of 12 antimicrobials. Notably, 30.1% of E. coli isolates were resistant to three or more antimicrobials. To minimize health risks associated with raw seafood consumption, more research is needed concerning the prevalence and antimicrobial resistance of fecal indicators.

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.

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.

Background Cognitive and emotional disturbances are common serious issues in patients with traumatic brain injury (TBI). However, predictors associated with neuropsychological functions were not consistent. Objective To investigate factors affecting cognition and emotion in patients with TBI, we evaluated executive function, memory, and emotion based on injury severity and lesion location. Methods Neuropsychological outcomes of 80 TBI patients were evaluated via Wisconsin Card Sorting Test (WCST), Color Trail Test (CTT), Controlled Oral Word Association Test (COWAT), Everyday Memory Questionnaire (EMQ), Geriatric Depression Scale (GDS), State-Trait Anxiety Inventory (STAI), and Agitated Behavior Scale (ABS). WCST, CTT, and COWAT assessed executive function; EMQ assessed everyday memory; and GDS, STAI, and ABS assessed emotion. Patients were categorized according to lateralization of lesion and existence of frontal lobe injury. Results Patients with longer duration of loss of consciousness (LOC) showed more severe deficits in everyday memory and agitated behaviors. The frontal lesion group showed poorer performance in executive function and higher agitation than the non-frontal lesion group. Patients with bilateral frontal lesion showed greater deficits in executive function and were more depressed than unilateral frontal lesion groups. Especially in those unilateral frontal lesion groups, right side frontal lesion group was worse on executive function than left side frontal lesion group. Conclusions Duration of LOC and lesion location are main parameters affecting executive function, everyday memory, and emotion in neuropsychological outcomes following TBI, suggesting that these parameters need to be considered for cognitive rehabilitation interventions.

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.

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.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. The mechanisms underlying PD remain to be fully elucidated, and research into treatments for this condition is ongoing. Recent advances in genetic research have shed light on the mechanisms underlying PD. In this study, we used PD and control mesenchymal stem cells (MSCs) obtained from adipose tissues to confirm the differences between groups at the cellular and molecular levels. The results revealed that in PD MSCs, cell viability was clearly lower, and the rate of cell senescence was higher compared to the controls. Next, to compare the gene expression in PD and control cells, transcriptome analysis was performed. Genes in pathways, including extracellular matrix (ECM) receptor interaction, P53 signaling, and focal adhesion, were down-regulated in PD. Among genes related to ECM receptor interaction, RELN gene expression was markedly decreased in PD cells, however, after being treated with recombinant Reelin protein, a significant increase in cell viability and a decrease in α-Synuclein aggregation and cell senescence were observed. In conclusion, Reelin affects PD by positively influencing the cell characteristics. Our findings will facilitate research into new treatments for PD.

Swallowing difficulties are a common complaint among patients with a variety of diseases. To address these concerns, a singing-enhanced swallowing protocol was constructed, and its differential benefits for two patient populations were investigated. Two patients with Parkinson’s disease (PD) and two patients with head and neck cancer (HNC) participated in this study. Each patient participated in 30-min individual sessions of a singing-enhanced swallowing protocol two times per week for 12 weeks. Following the intervention, laryngeal diadochokinesis and quality-of-life measurements were found to be higher in all four patients. However, the Videofluoroscopic Dysphagia Scale showed this improvement was associated with different swallowing tasks for each patient group. In addition, the maximum phonation time decreased for patients with HNC, while it increased for patients with PD. The findings support the use of a singing-enhanced swallowing protocol for patients whose swallowing difficulties are due to neurological or structural impairment. In addition, the study results suggest that different intervention components should be considered depending on the etiology of the patient’s swallowing difficulties.

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

Schwann cells (SCs) constitute a crucial element of the peripheral nervous system, by structurally supporting the formation of myelin and conveying vital trophic factors to the nervous system. However, the functions of SCs in developmental and regenerative stages remain unclear. Here, we investigated how optogenetic stimulation (OS) of SCs regulates their development. In SC monoculture, OS substantially enhanced SC proliferation and the number of BrdU+-S100ß+-SCs over time. In addition, OS also markedly promoted the expression of both Krox20 and myelin basic protein (MBP) in SC culture medium containing dBcAMP/NRG1, which induced differentiation. We found that the effects of OS are dependent on the intracellular Ca2+ level. OS induces elevated intracellular Ca2+ levels through the T-type voltage-gated calcium channel (VGCC) and mobilization of Ca2+ from both inositol 1,4,5-trisphosphate (IP3)-sensitive stores and caffeine/ryanodine-sensitive stores. Furthermore, we confirmed that OS significantly increased expression levels of both Krox20 and MBP in SC-motor neuron (MN) coculture, which was notably prevented by pharmacological intervention with Ca2+. Taken together, our results demonstrate that OS of SCs increases the intracellular Ca2+ level and can regulate proliferation, differentiation, and myelination, suggesting that OS of SCs may offer a new approach to the treatment of neurodegenerative disorders.