MultiSite Gateway Technology Is Useful for Donor DNA Plasmid Construction in CRISPR/Cas9-Mediated Knock-In System

The clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 method is a powerful tool for genome editing, by introducing a DNA double-strand break (DSB) at the specific site. The gene knock-out can be achieved by the deletion or insertion at the CRISPR/Cas9-mediated DSB site by error-prone nonhomologous end joining repair in targeted cells. However, the gene knock-in is still difficult as compared to the knock-out, because of the low efficiency of homology directed repair with donor DNA in cells. Therefore, to efficiently select the knock-in cells, we developed a complicated donor DNA plasmid containing an antibiotic-resistance gene, in addition to the knock-in sequence and the two homology arms. MultiSite Gateway technology is a useful tool for constructing this complicated plasmid. We describe the MultiSite Gateway technology and provide an overview of the DSB repair pathways to clarify the knock-out and knock-in methods by the CRISPR/Cas9 system

Locally embedded presages of global network bursts

Spontaneous, synchronous bursting of neural population is a widely observed phenomenon in nervous networks, which is considered important for functions and dysfunctions of the brain. However, how the global synchrony across a large number of neurons emerges from an initially non-bursting network state is not fully understood. In this study, we develop a new state-space reconstruction method combined with high-resolution recordings of cultured neurons. This method extracts deterministic signatures of upcoming global bursts in "local" dynamics of individual neurons during non-bursting periods. We find that local information within a single-cell time series can compare with or even outperform the global mean field activity for predicting future global bursts. Moreover, the inter-cell variability in the burst predictability is found to reflect the network structure realized in the non-bursting periods. These findings demonstrate the deterministic mechanisms underlying the locally concentrated early-warnings of the global state transition in self-organized networks

12-O-Tetradecanoylphorbol-13-acetate Induces Epstein–Barr Virus Reactivation via NF-κB and AP-1 as Regulated by Protein Kinase C and Mitogen-Activated Protein Kinase

AbstractSignaling pathway components mediating Epstein–Barr virus (EBV) reactivation by 12-O-tetradecanoylphorbol-13-acetate (TPA) were characterized in terms of induction and modification of specific transacting factors. The consequences of protein kinase C (PKC) activation by TPA in inhibiting inducible nitric oxide synthase (iNOS) mRNA expression were analyzed in the EBV-infected gastric epithelial cell line GT38. Spontaneous expression of the EBV BZLF1 gene product ZEBRA became undetectable upon long-term culturing of GT38 cells, while iNOS mRNA expression increased. In such cells the PKC inhibitors 1-(5-isoquinolinesulphonyl)-2,5-dimethylpiperazine (H7) and staurosporine inhibited TPA-induced expression of BZLF1 and BRLF1 and reversed TPA-mediated inhibition of iNOS gene expression. The mitogen-activated protein kinase inhibitor PD98059 inhibited TPA-induced BZLF1 expression. Electrophoretic mobility shift assays demonstrated that transcription factors NF-κB and AP-1 were also activated by TPA in a time-dependent manner. The TPA-induced NF-κB activation was inhibited by prior treatment of the cells with the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). TPA-induced BZLF1 expression was also inhibited by the treatment with PDTC. Northern blot analyses characterized changes in levels of the c-jun and junB expressions of the AP-1 family. These results show that TPA induces EBV reactivation via NF-κB and AP-1 and that PKC is an important mediator in regulating gene expression leading to EBV reactivation after TPA treatment of GT38 cells

Wireless optogenetics protects against obesity via stimulation of non-canonical fat thermogenesis.

Cold stimuli and the subsequent activation of β-adrenergic receptor (β-AR) potently stimulate adipose tissue thermogenesis and increase whole-body energy expenditure. However, systemic activation of the β3-AR pathway inevitably increases blood pressure, a significant risk factor for cardiovascular disease, and, thus, limits its application for the treatment of obesity. To activate fat thermogenesis under tight spatiotemporal control without external stimuli, here, we report an implantable wireless optogenetic device that bypasses the β-AR pathway and triggers Ca2+ cycling selectively in adipocytes. The wireless optogenetics stimulation in the subcutaneous adipose tissue potently activates Ca2+ cycling fat thermogenesis and increases whole-body energy expenditure without cold stimuli. Significantly, the light-induced fat thermogenesis was sufficient to protect mice from diet-induced body-weight gain. The present study provides the first proof-of-concept that fat-specific cold mimetics via activating non-canonical thermogenesis protect against obesity

Mitochondrial lipoylation integrates age-associated decline in brown fat thermogenesis.

Thermogenesis in brown adipose tissue (BAT) declines with age; however, what regulates this process remains poorly understood. Here, we identify mitochondria lipoylation as a previously unappreciated molecular hallmark of aged BAT in mice. Using mitochondrial proteomics, we show that mitochondrial lipoylation is disproportionally reduced in aged BAT through a post-transcriptional decrease in the iron-sulfur (Fe-S) cluster formation pathway. A defect in the Fe-S cluster formation by the fat-specific deletion of Bola3 significantly reduces mitochondrial lipoylation and fuel oxidation in BAT, leading to glucose intolerance and obesity. In turn, enhanced mitochondrial lipoylation by α-lipoic acid supplementation effectively restores BAT function in old mice, thereby preventing age-associated obesity and glucose intolerance. The effect of α-lipoic acids requires mitochondrial lipoylation via the Bola3 pathway and does not depend on the anti-oxidant activity of α-lipoic acid. These results open up the possibility to alleviate the age-associated decline in energy expenditure by enhancing the mitochondrial lipoylation pathway

Tumorigenesis of Epstein–Barr Virus-Positive Epithelial Cell Lines Derived from Gastric Tissues in the SCID Mouse

AbstractTo study the tumorigenesis of Epstein–Barr virus (EBV)-positive epithelial cell lines GT38 and GT39 derived from human gastric tissues, we inoculated these cells under the skin of severe combined immunodeficient (SCID) mice. The development of tumors was observed in each of the mice about 2 months after the inoculation. The tumors were diagnosed with undifferentiated carcinoma by hematoxylin/eosin staining. EBV-encoded small RNA1 was detected in the paraffin-embedded tumor sections. The tumor cells had human chromosome. The circular, but not linear, EBV DNA was detected in the tumors. The molecular sizes of EBV DNA termini were the same as that of the inoculated GT38 or GT39 cells. The expressions of EBV nuclear antigen 2 and latent membrane protein 1 reduced in the tumors. Transcripts of BamHI C and W promoters in latency III were detected in the tumors and the cultured cells in vitro. The tumor cells were passaged from one SCID mouse to other SCID mice and to cultures in vitro. This is the first evidence that the EBV-positive epithelial cell lines produced tumors in the SCID mouse

A Possible Protective Effect of the 'Cam Deformity' on Femoral Neck Fracture: The Relationship between Hip Morphology and the Types of Hip Fracture

We retrospectively evaluated the cases of 169 hip fracture patients, their previous fractures, and the contralateral hip joint’s morphology. A history of contralateral hip fracture was present in 23 patients (Contra group). The other patients had a unilateral hip fracture: a trochanteric fracture (Troch group, n=73) or a femoral neck fracture (Neck group, n=73). In the Troch and Neck groups, we used anteroposterior and cross-table axialview radiographs of the contralateral hip to evaluate the proximal femur’s anatomy. In the Contra group, the concordance rate between the first and second types of hip fracture was 65.2%, and the second hip fracture’s morphology indicated that the trochanteric fracture had a cam deformity in terms of the femoral head-neck ratio. The average alpha angle and femoral head-neck offset in the Troch group were significantly larger than those in the Neck group. In the Neck group, pistol-grip deformities of Arbeitsgemeinschaft für Osteosynthesefragen types B1 (subcapital), B2 (transcervical), and B3 (displaced) were observed in 42.1%, 75%, and 6% of cases, respectively. There was a smaller alpha angle and a larger femoral head-neck offset in the contralateral hip of femoral neck fractures; thus, the “cam deformity” may protect against femoral neck fractures

Non-coplanar spin structure in a metallic thin film of triangular lattice antiferromagnet CrSe

Tajima Y., Shiogai J., Ueda K., et al. APL Materials, 12, 041112 (2024); licensed under a Creative Commons Attribution (CC BY) license.An antiferromagnetic metal with a two-dimensional triangular network offers a unique playground of intriguing magneto-transport properties and functionalities stemming from the interplay between conducting electrons and intricate magnetic phases. A NiAs-type CrSe is one of the candidates owing to alternate stackings of Cr and Se triangular atomic networks in its crystal structure. While the fabrication of CrSe thin films is indispensable to develop functional devices, studies on its thin-film properties have been limited to date due to the lack of metallic samples. Here, we report on the realization of metallic conductivities of CrSe thin films, which allows us to investigate their intrinsic magneto-transport properties. The metallic sample exhibits a co-occurrence of weak ferromagnetism with perpendicular magnetic anisotropy and antiferromagnetic behavior, indicating the presence of non-coplanar spin structures. In addition, control of the polarity and tilting angle of the non-coplanar spin structure is accomplished by a sign of cooling magnetic fields. The observed non-coplanar spin structure, which can be a source of emergent magnetic field acting on the conducting electrons, highlights the high potential of the triangular lattice antiferromagnet and provides a unique platform for functional thin-film devices composed of NiAs-type derivative Cr chalcogenides and pnictides