CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic ...loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
The first analytic topologically non-trivial solutions in the (3 + 1)-dimensional gauged non-linear sigma model representing multi-solitons at finite volume with manifest ordered structures ...generating their own electromagnetic field are presented. The complete set of seven coupled non-linear field equations of the gauged non-linear sigma model together with the corresponding Maxwell equations are reduced in a self-consistent way to just one linear Schrodinger-like equation in two dimensions. The corresponding two dimensional periodic potential can be computed explicitly in terms of the solitons profile. The present construction keeps alive the topological charge of the gauged solitons. Both the energy density and the topological charge density are periodic and the positions of their peaks show a crystalline order. These solitons describe configurations in which (most of) the topological charge and total energy are concentrated within three-dimensional tube-shaped regions. The electric and magnetic fields vanish in the center of the tubes and take their maximum values on their surface while the electromagnetic current is contained within these tube-shaped regions. Electromagnetic perturbations of these families of gauged solitons are shortly discussed.
Although intravenous administration of mesenchymal stem cells (MSCs) is effective for experimental stroke, low engraftment and the limited functional capacity of transplanted cells are critical ...hurdles for clinical applications. C–C motif chemokine ligand 2 (CCL2) is associated with neurological repair after stroke and delivery of various cells into the brain via CCL2/CCR2 (CCL2 receptor) interaction. In this study, after CCL2-overexpressing human umbilical cord-derived MSCs (hUC-MSCs) were intravenously transplanted with mannitol in rats with middle cerebral arterial occlusion, we compared the differences between four different treatment groups: mannitol + CCL2-overexpressing hUC-MSCs (CCL2-MSC), mannitol + naïve hUC-MSCs (M-MSC), mannitol only, and control. At four-weeks post-transplantation, the CCL2-MSC group showed significantly better functional recovery and smaller stroke volume relative to the other groups. Additionally, we observed upregulated levels of CCR2 in acute ischemic brain and the increase of migrated stem cells into these areas in the CCL2-MSC group relative to the M-MSC. Moreover, the CCL2-MSC group displayed increased angiogenesis and endogenous neurogenesis, decreased neuro-inflammation but with increased healing-process inflammatory cells relative to other groups. These findings indicated that CCL2-overexpressing hUC-MSCs showed better functional recovery relative to naïve hUC-MSCs according to the increased migration of these cells into brain areas of higher CCR2 expression, thereby promoting subsequent endogenous brain repair.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder of the central nervous system (CNS) that is defined by a CAG expansion in exon 1 of the huntingtin gene leading to the ...production of mutant huntingtin (mHtt). To date, the disease pathophysiology has been thought to be primarily driven by cell-autonomous mechanisms, but, here, we demonstrate that fibroblasts derived from HD patients carrying either 72, 143 and 180 CAG repeats as well as induced pluripotent stem cells (iPSCs) also characterized by 143 CAG repeats can transmit protein aggregates to genetically unrelated and healthy host tissue following implantation into the cerebral ventricles of neonatal mice in a non-cell-autonomous fashion. Transmitted mHtt aggregates gave rise to both motor and cognitive impairments, loss of striatal medium spiny neurons, increased inflammation and gliosis in associated brain regions, thereby recapitulating the behavioural and pathological phenotypes which characterizes HD. In addition, both in vitro work using co-cultures of mouse neural stem cells with 143 CAG fibroblasts and the SH-SY5Y human neuroblastoma cell line as well as in vivo experiments conducted in newborn wild-type mice suggest that exosomes can cargo mHtt between cells triggering the manifestation of HD-related behaviour and pathology. This is the first evidence of human-to-mouse prion-like propagation of mHtt in the mammalian brain; a finding which will help unravel the molecular bases of HD pathology as well as to lead to the development of a whole new range of therapies for neurodegenerative diseases of the CNS.
Objectives
Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic ...development, has long been an issue. OCT4 has a species‐specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4‐related pluripotency networks and second‐cell fate decisions.
Materials and Methods
To determine the functions of OCT4 in lineage specification and embryo proliferation, loss‐ and gain‐of–function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real‐time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes.
Results
In OCT4‐targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real‐time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4‐targeted blastocysts, but trophoblast‐related genes were increased. In OCT4‐overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real‐time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased.
Conclusion
Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.
We analyzed the phenotypes of OCT4 knockout and OCT4 overexpressing preimplantation porcine embryos to understand the function of OCT4, one of the core transcription factors. In the OCT4 targeted embryos, intact inner cell mass (ICM) was not formed, and the expression of pluripotency‐related genes and proliferation‐related genes was decreased. In OCT4 overexpression embryos, the expression of the primitive endoderm marker SOX17 was decreased and the expression of epiblast marker genes increased. These results suggest that OCT4 is involved in both cell fate decisions in porcine preimplantation embryos and also affects cell proliferation.
Genetic neuromuscular diseases are clinically and genetically heterogeneous genetic disorders that primarily affect the peripheral nerves, muscles, and neuromuscular junctions. This study aimed to ...identify pathogenic variants, calculate carrier frequency, and predict the genetic prevalence of autosomal recessive neuromuscular diseases (AR-NMDs). We selected 268 AR-NMD genes and analyzed their genetic variants sourced from the gnomAD database. After identifying the pathogenic variants using an algorithm, we calculated the carrier frequency and predicted the genetic prevalence of AR-NMDs. In total, 10,887 pathogenic variants were identified, including 3848 literature verified and 7039 manually verified variants. In the global population, the carrier frequency of AR-NMDs is 32.9%, with variations across subpopulations ranging from 22.4% in the Finnish population to 36.2% in the non-Finnish European population. The predicted genetic prevalence of AR-NMDs was estimated to be 24.3 cases per 100,000 individuals worldwide, with variations across subpopulations ranging from 26.5 to 41.4 cases per 100,000 individuals in the Latino/Admixed American and the Ashkenazi Jewish populations, respectively. The AR-NMD gene with the highest carrier frequency was GAA (1.3%) and the variant with the highest allele frequency was c.-32-13 T>G in GAA with 0.0033 in the global population. Our study revealed a higher-than-expected frequency of AR-NMD carriers, constituting approximately one-third of the global population, highlighting ethnic heterogeneity in genetic susceptibility.
Objectives
Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell ...mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.
Materials and methods
To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.
Results
Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.
Conclusions
Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.
We analysed the phenotypes of SOX2 knockout and SOX2‐overexpressing pre‐implantation porcine embryos to understand the function of SOX2, one of the core transcription factors. In SOX2‐targeted embryos, total cell number decreased, and expression of pluripotent‐related genes and proliferation‐related genes decreased. Loss‐ and gain‐of‐function analysis revealed that SOX2 regulates total cell number and expression of pluripotent‐related genes and proliferation‐related genes. These results indicate that SOX2 plays important role in cell proliferation and ICM formation in porcine pre‐implantation embryos.
Two analytic examples of globally regular non-Abelian gravitating solitons in the Einstein–Yang–Mills–Higgs theory in (3 + 1)-dimensions are presented. In both cases, the space-time geometries are of ...the Nariai type and the Yang–Mills field is completely regular and of meron type (namely, proportional to a pure gauge). However, while in the first family (type I)
X
0
=
1
/
2
(as in all the known examples of merons available so far) and the Higgs field is trivial, in the second family (type II)
X
0
=
1
/
2
is not 1/2 and the Higgs field is non-trivial. We compare the entropies of type I and type II families determining when type II solitons are favored over type I solitons: the VEV of the Higgs field plays a crucial role in determining the phases of the system. The Klein–Gordon equation for test scalar fields coupled to the non-Abelian fields of the gravitating solitons can be written as the sum of a two-dimensional D’Alembert operator plus a Hamiltonian which has been proposed in the literature to describe the four-dimensional Quantum Hall Effect (QHE): the difference between type I and type II solutions manifests itself in a difference between the degeneracies of the corresponding energy levels.
To identify genetic factors influencing quantitative traits of biomedical importance, we conducted a genome-wide association study in 8,842 samples from population-based cohorts recruited in Korea. ...For height and body mass index, most variants detected overlapped those reported in European samples. For the other traits examined, replication of promising GWAS signals in 7,861 independent Korean samples identified six previously unknown loci. For pulse rate, signals reaching genome-wide significance mapped to chromosomes 1q32 (rs12731740, P = 2.9 x 10(-9)) and 6q22 (rs12110693, P = 1.6 x 10(-9)), with the latter approximately 400 kb from the coding sequence of GJA1. For systolic blood pressure, the most compelling association involved chromosome 12q21 and variants near the ATP2B1 gene (rs17249754, P = 1.3 x 10(-7)). For waist-hip ratio, variants on chromosome 12q24 (rs2074356, P = 7.8 x 10(-12)) showed convincing associations, although no regional transcript has strong biological candidacy. Finally, we identified two loci influencing bone mineral density at multiple sites. On chromosome 7q31, rs7776725 (within the FAM3C gene) was associated with bone density at the radius (P = 1.0 x 10(-11)), tibia (P = 1.6 x 10(-6)) and heel (P = 1.9 x 10(-10)). On chromosome 7p14, rs1721400 (mapping close to SFRP4, a frizzled protein gene) showed consistent associations at the same three sites (P = 2.2 x 10(-3), P = 1.4 x 10(-7) and P = 6.0 x 10(-4), respectively). This large-scale GWA analysis of well-characterized Korean population-based samples highlights previously unknown biological pathways.
Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used ...in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntington's disease (HD) carrying 72 CAG repeats (HD-iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD-iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD-iPSC-derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly, during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD-iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD-iPSC will serve as useful tools to study HD pathology and develop novel therapeutics.
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