Dormant state of quiescent neural stem cells links Shank3 mutation to autism development.

Autism spectrum disorders (ASDs) are common neurodevelopmental disorders characterized by deficits in social interactions and communication, restricted interests, and repetitive behaviors. Despite extensive study, the molecular targets that control ASD development remain largely unclear. Here, we report that the dormancy of quiescent neural stem cells (qNSCs) is a therapeutic target for controlling the development of ASD phenotypes driven by Shank3 deficiency. Using single-cell RNA sequencing (scRNA-seq) and transposase accessible chromatin profiling (ATAC-seq), we find that abnormal epigenetic features including H3K4me3 accumulation due to up-regulation of Kmt2a levels lead to increased dormancy of qNSCs in the absence of Shank3. This result in decreased active neurogenesis in the Shank3 deficient mouse brain. Remarkably, pharmacological and molecular inhibition of qNSC dormancy restored adult neurogenesis and ameliorated the social deficits observed in Shank3-deficient mice. Moreover, we confirmed restored human qNSC activity rescues abnormal neurogenesis and autism-like phenotypes in SHANK3-targeted human NSCs. Taken together, our results offer a novel strategy to control qNSC activity as a potential therapeutic target for the development of autism.

Anticancer effect of locally applicable aptamer-conjugated gemcitabine-loaded atelocollagen patch in pancreatic cancer patient-derived xenograft models.

We investigated the anticancer effect of the aptamer-conjugated gemcitabine-loaded atelocollagen patch in a pancreatic cancer patient-derived xenograft (PDX) model to propose a future potential adjuvant surgical strategy during curative pancreatic resection for pancreatic cancer. A pancreatic cancer PDX model was established. Animals were grouped randomly into a no-treatment control group; treatment group treated with intraperitoneal gemcitabine injection (IP-GEM) or aptamer-conjugated gemcitabine (APT:GEM); and transplant with three kinds of patches: atelocollagen-aptamer-gemcitabine (patch I), atelocollagen-inactive aptamer-gemcitabine (patch II), and atelocollagen-gemcitabine (patch III). Tumor volumes and response were evaluated based on histological analysis by H&E staining and Immunohistochemistry (IHC) was performed. Anticancer therapy-related toxicity was evaluated by hematologic findings. The patch I group showed the most significant reduction of tumor growth rate, compared with the no-treatment group (p < 0.05). However, other treatment groups were not found to show significant reduction in tumor growth rate (0.05 < p < 0.1). There was no microscopic evidence suggesting potential toxicity, such as inflammation, nor necrotic changes in liver, lung, kidney, and spleen tissue. In addition, no leukopenia, anemia, or neutropenia was observed in the patch I group. This implantable aptamer-drug conjugate system is thought to be a new surgical strategy to augment the oncologic significance of margin-negative resection in treating pancreatic cancer in near future.

Activation of melatonin receptor 1 by CRISPR-Cas9 activator ameliorates cognitive deficits in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of neurotoxic beta-amyloid (Aß) in the brain. Melatonin receptors have been reported to associate with aging and AD, and their expression decreased with the progression of AD. As an alternative to AD treatment, overexpression of melatonin receptors may lead to melatonin-like effects to treat alleviate the symptoms of AD. Here, we successfully activated the type 1 melatonin receptor (Mt1) in vivo brain using a Cas9 activator as a novel AD therapeutic strategy. The Cas9 activator efficiently activated the endogenous Mt1 gene in the brain. Activation of Mt1 via Cas9 activators modulated anti-amyloidogenic and anti-inflammatory roles in 5xFAD AD mice brain. Moreover, activation of Mt1 with the CRISPR/Cas9 activator improved cognitive deficits in an AD model. These results demonstrated the therapeutic potential of melatonin receptor activation via CRISPR/Cas9 activator for AD.

Generation of Integration-Free Induced Neurons Using Graphene Oxide-Polyethylenimine.

Direct conversion of somatic cells into induced neurons (iNs) without inducing pluripotency has great therapeutic potential for treating central nervous system diseases. Reprogramming of somatic cells to iNs requires the introduction of several factors that drive cell-fate conversion, and viruses are commonly used to deliver these factors into somatic cells. However, novel gene-delivery systems that do not integrate transgenes into the genome are required to generate iNs for safe human clinical applications. In this study, it is investigated whether graphene oxide-polyethylenimine (GO-PEI) complexes are an efficient and safe system for messenger RNA delivery for direct reprogramming of iNs. The GO-PEI complexes show low cytotoxicity, high delivery efficiency, and directly converted fibroblasts into iNs without integrating factors into the genome. Moreover, in vivo transduction of reprogramming factors into the brain with GO-PEI complexes facilitates the production of iNs that alleviated Parkinson's disease symptoms in a mouse model. Thus, the GO-PEI delivery system may be used to safely obtain iNs and could be used to develop direct cell reprogramming-based therapies for neurodegenerative diseases.

Comparison of Supraclavicular and Infraclavicular Brachial Plexus Block: A Systemic Review of Randomized Controlled Trials.

BACKGROUND: Supraclavicular (SC) and infraclavicular (IC) brachial plexus block (BPB) are commonly used for upper extremity surgery. Recent clinical studies have compared the effect of SC- and IC-BPB, but there have been controversies over spread of sensory blockade in each of the 4 peripheral nerve branches of brachial plexus. METHODS: This study included a systemic review, using the Medline and EMBASE database from their inceptions through March 2016. Randomized controlled trials (RCTs) comparing SC- and IC-BPB were included. The prespecified primary outcome was the incidences of incomplete sensory blockade in each of the 4 terminal nerve branches of brachial plexus. Secondary outcome included the incidence of successful blockade, performance time, onset of sensory block, duration of analgesia, and complication rates. RESULTS: Ten RCTs involving 676 patients were included. Pooled analyses showed the incidence of incomplete block at 30 minutes in radial nerve territory was significantly higher in IC-BPB, favoring SC-BPB (risk ratio 0.39; 95% confidence interval [0.17-0.88], P = .02, I = 0%). However, subgroup analysis according to the number of injections of IC-BPB showed that double or triple injections IC-BPB yielded no difference in the incomplete radial block. Furthermore, the incidence of incomplete ulnar block at 30 minutes was significantly lower in IC-BPB when using double or triple injection IC-BPB. There was no difference in the secondary outcomes between SC- and IC-BPB groups, with the exception of complication rates. The incidence of paresthesia/pain on local anesthetic injection, phrenic nerve palsy, and Horner syndrome was significantly higher in the SC group, favoring IC-BPB. CONCLUSIONS: This meta-analysis demonstrated that IC-BPB showed a significantly high incidence of incomplete radial nerve sensory block at 30 minutes, which may be avoided by double or triple injection. Furthermore, IC-BPB with multiple injection technique showed significantly lower incidence of incomplete ulnar block than SC-BPB. There were no differences in the incidence of successful blockade, block onset, and duration of analgesia between SC- and IC-BPB. Procedure-related paresthesia/pain and adjacent nerve-related complications were more frequent in SC-BPB. However, because of the small sample size, publication bias remains a concern when interpreting our results. Further studies with sufficient sample size and reporting large number of outcomes are required.

Optimal level of the reference transducer for central venous pressure and pulmonary artery occlusion pressure monitoring in supine, prone, and sitting position.

To guarantee accurate measurement of central venous pressure (CVP) or pulmonary artery occlusion pressure (PAOP), proper positioning of a reference transducer is a prerequisite. We investigated ideal transducer levels in supine, prone, and sitting position in adults. Chest computed tomography images of 113 patients, taken in supine or prone position were reviewed. For supine position, distances between the back and the uppermost blood level of both atria and their ratios to the largest anteroposterior (AP) diameter of thorax were calculated. For prone position, same distances and ratios were calculated from the anterior chest. For sitting position, distances between the mid-sternoclavicular joint and the most cephalad blood level of both atria and their ratios to the sternal length were calculated. The ratio of the uppermost blood level of right atrium (RA) and left atrium (LA) to the largest AP diameter of thorax was 0.81 ± 0.04 and 0.59 ± 0.03 from the back in supine position. That calculated from the anterior chest in prone position was 0.54 ± 0.03 and 0.46 ± 0.03. The ratio of the most cephalad blood level of RA and LA to the sternal length was 0.70 ± 0.10 and 0.68 ± 0.09 from the mid-sternoclavicular joint in sitting position, which corresponded to the upper border of 4th rib. Optimal CVP transducer levels are at four-fifths of the AP diameter of thorax in supine position, at a half of that in prone position, and at upper border of the 4th sternochondral joint in sitting position. PAOP transducer levels are similar in prone and sitting position, except for supine position which is at three-fifths of the AP diameter of thorax.

The lack of relationship between intracranial pressure and cerebral ventricle indices based on brain computed tomography in patients undergoing ventriculoperitoneal shunt.

BACKGROUND: In this study we investigated whether cerebral ventricle indices based on brain computed tomography (CT) scans are reliable for predicting intracranial pressure (ICP) in hydrocephalic patients. METHODS: Electronic medical records of 221 patients undergoing ventriculoperitoneal shunt due to hydrocephalus were retrospectively reviewed. Cerebral ventricle indices including Evans' index, third ventricle index, cella media index, and ventricular score were calculated from transverse diameters measured at various levels on preoperative brain CT scans. ICP was considered as CSF opening pressure. Patients were categorized into three groups: communicating hydrocephalus, non-communicating hydrocephalus, and normal pressure hydrocephalus (NPH). The non-communicating hydrocephalus group was further divided according to the obstruction site; aqueduct, fourth ventricle outlet, third ventricle, and the foramen of Monro. The primary endpoint was the extent of the correlation between cerebral ventricle indices and ICP in each hydrocephalus group. RESULTS: No cerebral ventricle index correlated with ICP in patients with communicating hydrocephalus (n = 113) and NPH (n = 62). In the non-communicating hydrocephalus group (n = 46), only the third ventricle index revealed moderate negative correlation with ICP (r = -0.395, p < 0.01). In subgroup analyses, the third ventricle index showed a strong negative relationship with ICP only in patients with the third ventricle obstruction (r = -0.779, p < 0.05). CONCLUSIONS: In this study we showed that although an inverse correlation existed between ICP and the third ventricle index only in patients with non-communicating hydrocephalus due to obstruction of the third ventricle, cerebral ventricle indices based on brain CT scan were non-reliable predictors of ICP in hydrocephalic patients.

Development of a modified straw method for vitrification of in vitro-produced bovine blastocysts and various genes expression in between the methods.

This study evaluated a modified plastic straw loading method for vitrification of in vitro-produced bovine blastocysts. A modified straw was used with a depressed area on its inner surface to which embryos attach. In vitro-produced blastocysts were randomly assigned into three groups: (i) blastocysts attached to the inner surface of a plastic straw (aV), (ii) blastocysts attached to the inner surface of a modified plastic straw (maV), and (iii) non-vitrified blastocysts (control). The recovery rates were not significantly different between aV and maV groups (95.8% vs. 94.3%). The post-thaw survival rate did not significantly differ between aV and maV groups (86.4% vs. 88.2%). The total cell numbers of blastocyst was higher in control than in aV and maV groups (142 ± 21.8 vs. 117 ± 29.7 and 120 ± 25.2; P < 0.05), but not significantly differ between aV and maV groups. The mRNA levels of pro-apoptosis related genes Bax and Caspase-3 were higher in aV and maV than in control (P < 0.05). By contrast, the mRNA levels of anti-apoptotic genes Bcl-2 and Mcl-1 and of antioxidant-related genes MnSOD and Prdx5 were lower in aV and maV than in control (P < 0.05). Confocal microscopy analysis of Golgi apparatus and mitochondria showed that the fluorescence intensity of Golgi apparatus and mitochondria was higher in control than in aV and maV groups. In conclusion, both aV and maV methods can be used to successfully vitrify IVP blastocysts, with maV method to be preferable because of its easiness in embryo loading.

The Effect of κ-Carrageenan on Porcine Sperm Cryo-Survival.

κ-Carrageenan is a sulfated polysaccharide from red seaweed with substantial antioxidant activities. This study aimed to investigate the effect of κ-Carrageenan treatment on frozen-thawed (FT) porcine semen quality. Therefore, the spermatozoa were diluted and cryopreserved in a freezing extender supplemented with 0 (control), 0.2, 0.4, 0.6, and 0.8 mg/mL κ-Carrageenan. Sperm kinematics were assessed immediately after thawing (AT) and post-incubation for 120 min. The viability, acrosome integrity, lipid peroxidation, mitochondrial membrane potential (MMP), and intracellular caspase activity were measured AT. The results indicated that 0.2 mg/mL κ-Carrageenan increased total and progressive motility AT and post-incubation for 120 min (p < 0.05). Moreover, the viable sperm percentage and MMP after 0.2 mg/mL treatment were higher than those after control and other κ-Carrageenan concentration treatments. The proportion of acrosome-intact spermatozoa was significantly higher after 0.2 and 0.4 mg/mL κ-Carrageenan treatment than that after control and other κ-Carrageenan concentration treatments. The intracellular caspase activity was not significantly different among the experimental groups. However, the MDA concentration after 0.2 mg/mL κ-Carrageenan treatment was lower (p < 0.05) than that after the control treatment. Taken together, adding κ-Carrageenan to the porcine semen freezing extender improved the FT sperm quality mainly by influencing membrane stability and protecting against oxidative stress.

Single-cell RNA-sequencing identifies disease-associated oligodendrocytes in male APP NL-G-F and 5XFAD mice.

Alzheimer's disease (AD) is associated with progressive neuronal degeneration as amyloid-beta (Aß) and tau proteins accumulate in the brain. Glial cells were recently reported to play an important role in the development of AD. However, little is known about the role of oligodendrocytes in AD pathogenesis. Here, we describe a disease-associated subpopulation of oligodendrocytes that is present during progression of AD-like pathology in the male AppNL-G-F and male 5xFAD AD mouse brains and in postmortem AD human brains using single-cell RNA sequencing analysis. Aberrant Erk1/2 signaling was found to be associated with the activation of disease-associated oligodendrocytes (DAOs) in male AppNL-G-F mouse brains. Notably, inhibition of Erk1/2 signaling in DAOs rescued impaired axonal myelination and ameliorated Aß-associated pathologies and cognitive decline in the male AppNL-G-F AD mouse model.

(E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol alleviates inflammatory responses in LPS-induced mice liver sepsis through inhibition of STAT3 phosphorylation.

Sepsis is a life-threatening disease with limited treatment options, and the inflammatory process represents an important factor affecting its progression. Many studies have demonstrated the critical roles of signal transducer and activator of transcription 3 (STAT3) in sepsis pathophysiology and pro-inflammatory responses. Inhibition of STAT3 activity may therefore represent a promising treatment option for sepsis. We here used a mouse model to demonstrate that (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) treatment prevented the liver sepsis-related mortality induced by 30 mg/kg lipopolysaccharide (LPS) treatment and reduced LPS-induced increase in alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels, all of which are markers of liver sepsis progression. These recovery effects were associated with decreased LPS-induced STAT3, p65, and JAK1 phosphorylation and proinflammatory cytokine (interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha) level; expression of cyclooxygenase-2 and induced nitric oxide synthase were also reduced by MMPP. In an in vitro study using the normal liver cell line THLE-2, MMPP treatment prevented the LPS-induced increase of STAT3, p65, and JAK1 phosphorylation and inflammatory protein expression in a dose-dependent manner, and this effect was enhanced by combination treatment with MMPP and STAT3 inhibitor. The results clearly indicate that MMPP treatment prevents LPS-induced mortality by inhibiting the inflammatory response via STAT3 activity inhibition. Thus, MMPP represents a novel agent for alleviating LPS-induced liver sepsis.

CRISPR/dCas9-Dnmt3a-mediated targeted DNA methylation of APP rescues brain pathology in a mouse model of Alzheimer's disease.

BACKGROUND: Aberrant DNA methylation patterns have been observed in neurodegenerative diseases, including Alzheimer's disease (AD), and dynamic changes in DNA methylation are closely associated with the onset and progression of these diseases. Particularly, hypomethylation of the amyloid precursor protein gene (APP) has been reported in patients with AD. METHODS: In this study, we used catalytically inactivated Cas9 (dCas9) fused with Dnmt3a for targeted DNA methylation of APP, and showed that the CRISPR/dCas9-Dnmt3a-mediated DNA methylation system could efficiently induce targeted DNA methylation of APP both in vivo and in vitro. RESULTS: We hypothesized that the targeted methylation of the APP promoter might rescue AD-related neuronal cell death by reducing APP mRNA expression. The cultured APP-KI mouse primary neurons exhibited an altered DNA-methylation pattern on the APP promoter after dCas9-Dnmt3a treatment. Likewise, the APP mRNA level was significantly reduced in the dCas9-Dnmt3a-treated wild-type and APP-KI mouse primary neurons. We also observed decreased amyloid-beta (Aß) peptide level and Aß42/40 ratio in the dCas9-Dnmt3a-treated APP-KI mouse neurons compared to the control APP-KI mouse neurons. In addition, neuronal cell death was significantly decreased in the dCas9-Dnmt3a-treated APP-KI mouse neurons. Furthermore, the in vivo methylation of APP in the brain via dCas9-Dnmt3a treatment altered Aß plaques and attenuated cognitive and behavioral impairments in the APP-KI mouse model. CONCLUSIONS: These results suggest that the targeted methylation of APP via dCas9-Dnmt3a treatment can be a potential therapeutic strategy for AD.

In vivo therapeutic genome editing via CRISPR/Cas9 magnetoplexes for myocardial infarction.

CRISPR/Cas9-mediated gene-editing technology has gained attention as a new therapeutic method for intractable diseases. However, the use of CRISPR/Cas9 for cardiac conditions such as myocardial infarction remains challenging due to technical and biological barriers, particularly difficulties in delivering the system and targeting genes in the heart. In the present study, we demonstrated the in vivo efficacy of the CRISPR/Cas9 magnetoplexes system for therapeutic genome editing in myocardial infarction. First, we developed CRISPR/Cas9 magnetoplexes that magnetically guided CRISPR/Cas9 system to the heart for efficient in vivo therapeutic gene targeting during heart failures. We then demonstrated that the in vivo gene targeting of miR34a via these CRISPR/Cas9 magnetoplexes in a mouse model of myocardial infarction significantly improved cardiac repair and regeneration to facilitate improvements in cardiac function. These results indicated that CRISPR/Cas9 magnetoplexes represent an effective in vivo therapeutic gene-targeting platform in the myocardial infarction of heart, and that this strategy may be applicable for the treatment of a broad range of cardiac failures.

Transcriptional activation with Cas9 activator nanocomplexes rescues Alzheimer's disease pathology.

CRISPR/Cas9-mediated gene activation is a potential therapeutic strategy that does not induce double-strand break (DSB) DNA damage. However, in vivo gene activation via a Cas9 activator remains a challenge, currently limiting its therapeutic applications. We developed a Cas9 activator nanocomplex that efficiently activates an endogenous gene in the brain in vivo, suggesting its possible application in novel therapeutics. We demonstrated a potential treatment application of the Cas9 activator nanocomplex by activating Adam10 in the mouse brain without introducing insertions and deletions (inDels). Remarkably, in vivo activation of Adam10 with the Cas9 activator nanocomplex improved cognitive deficits in an Alzheimer's disease (AD) mouse model. These results demonstrate the therapeutic potential of Cas9 activator nanocomplexes for a wide range of neurological diseases.

Rad50 mediates DNA demethylation to establish pluripotent reprogramming.

DNA demethylation is characterized by the loss of methyl groups from 5-methylcytosine, and this activity is involved in various biological processes in mammalian cell development and differentiation. In particular, dynamic DNA demethylation in the process of somatic cell reprogramming is required for successful iPSC generation. In the present study, we reported the role of Rad50 in the DNA demethylation process during somatic cell reprogramming. We found that Rad50 was highly expressed in pluripotent stem cells and that Rad50 regulated global DNA demethylation levels. Importantly, the overexpression of Rad50 resulted in the enhanced efficiency of iPSC generation via increased DNA demethylation, whereas Rad50 knockdown led to DNA hypermethylation, which suppressed somatic cell reprogramming into iPSCs. Moreover, we found that Rad50 associated with Tet1 to facilitate the DNA demethylation process in pluripotent reprogramming. Therefore, our findings highlight the novel role of Rad50 in the DNA demethylation process during somatic cell reprogramming.

Valproic Acid Significantly Improves CRISPR/Cas9-Mediated Gene Editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has emerged as a powerful technology, with the potential to generate transgenic animals. Particularly, efficient and precise genetic editing with CRISPR/Cas9 offers immense prospects in various biotechnological applications. Here, we report that the histone deacetylase inhibitor valproic acid (VPA) significantly increases the efficiency of CRISPR/Cas9-mediated gene editing in mouse embryonic stem cells and embryos. This effect may be caused through globally enhanced chromatin accessibility, as indicate by histone hyperacetylation. Taken together, our results suggest that VPA can be used to increase the efficacy of CRISPR/Cas9 in generating transgenic systems.

Modelling neurodegenerative diseases with 3D brain organoids.

Neurodegenerative diseases are incurable and debilitating conditions characterized by the deterioration of brain function. Most brain disease models rely on human post-mortem brain tissue, non-human primate tissue, or in vitro two-dimensional (2D) experiments. Resource limitations and the complexity of the human brain are some of the reasons that make suitable human neurodegenerative disease models inaccessible. However, recently developed three-dimensional (3D) brain organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells and induced PSCs, may provide suitable models for the study of the pathological features of neurodegenerative diseases. In this review, we provide an overview of existing 3D brain organoid models and discuss recent advances in organoid technology that have increased our understanding of brain development. Moreover, we explain how 3D organoid models recapitulate aspects of specific neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease, and explore the utility of these models, for therapeutic applications.

Effects of a hypomagnetic field on DNA methylation during the differentiation of embryonic stem cells.

It has been reported that hypomagnetic fields (HMFs) have a negative influence on mammalian physiological functions. We previously reported that HMFs were detrimental to cell fate changes during reprogramming into pluripotency. These studies led us to investigate whether HMFs affect cell fate determination during direct differentiation. Here, we found that an HMF environment attenuates differentiation capacity and is detrimental to cell fate changes during the in vitro differentiation of embryonic stem cells (ESCs). Moreover, HMF conditions cause abnormal DNA methylation through the dysregulation of DNA methyltransferase3b (Dnmt3b) expression, eventually resulting in incomplete DNA methylation during differentiation. Taken together, these results suggest that an appropriate electromagnetic field (EMF) environment may be essential for favorable epigenetic remodeling during cell fate determination via differentiation.