Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing

Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse

h-pruneは大腸癌肝転移患者の予後と上皮間葉転換に関連している

内容の要旨 , 審査の要旨広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

Adsorption of Laminin on Hydroxyapatite and Alumina and the MC3T3-E1 Cell Response

Artificial hydroxyapatite (HAp) is osteoconductive, but the mechanism is still unclear. It is likely that some serum proteins are adsorbed onto HAp and influence its osteoconductivity. We investigated the adsorption behavior of laminin (LN), which was isolated from murine Engelbreth–Holm–Swarm sarcoma, onto HAp and compared it with nonosteoconductive alpha-type alumina (α-Al2O3). Cell adhesion, spreading, and proliferation on native and LN-adsorbed discs of HAp or α-Al2O3 were examined using murine MC3T3-E1 osteoblastic cells. A larger amount of LN adsorbed onto HAp than α-Al2O3 despite the electrostatic repulsion between LN and HAp, suggesting the specific adsorption of LN onto HAp. The LN adsorbed onto HAp remarkably enhanced initial attachment and spreading of MC3T3-E1 cells, but subsequent proliferation of MC3T3-E1 cells was influenced by the type of material rather than LN adsorption. These fundamental findings imply that LN adsorbed on HAp could trigger osteoconductivity in vivo, aiding in the development of novel biomaterials that specifically adsorb LN and effectively enhance cell attachment and spreading

Fibronectin Adsorption on Osteoconductive Hydroxyapatite and Non-osteoconductive α-alumina

The osteoconductivity mechanism of hydroxyapatite (HAp) has not been elucidated. It is hypothesized that specific proteins adsorb on HAp, promoting its osteoconductivity. To verify this hypothesis, we compared the adsorption behavior of fibronectin (Fn) on HAp powder and on α-alumina (α-Al2O3) powder, a material with no osteoconductivity. More Fn adsorbed on α-Al2O3 than on HAp, irrespective of the Fn concentration, and there was no significant difference in the secondary structure of Fn adsorbed on HAp and α-Al2O3. Further, it is possible that Fn did not adsorb on HAp and α-Al2O3 through the Arg-Gry-Asp motif of Fn. The amount of Fn adsorbed on HAp oriented to the a(b)-axis with very little decrease in carbonate and the adsorbed Fn had a smaller α-helix structure content. The results suggest that the secondary and/or higher-order structure rather than the amount of adsorbed Fn might affect the osteoconductivity of HAp, which might be electrostatically controlled by the crystal face orientation and/or carbonate content of HAp, although this should be confirmed by a cell culture test in the future

Adsorption characteristics of bovine serum albumin onto alumina with a specific crystalline structure

Bone cement containing alumina particles with a specific crystalline structure exhibits the ability to bond with bone. These particles (AL-P) are mainly composed of delta-type alumina (δ-Al2O3). It is likely that some of the proteins present in the body environment are adsorbed onto the cement and influence the expression of its bioactivity. However, the effect that this adsorption of proteins has on the bone-bonding mechanism of bone cement has not yet been elucidated. In this study, we investigated the characteristics of the adsorption of bovine serum albumin (BSA) onto AL-P and compared them with those of its adsorption onto hydroxyapatite (HA), which also exhibits bone-bonding ability, as well as with those of adsorption onto alpha-type alumina (α-Al2O3), which does not bond with bone. The adsorption characteristics of BSA onto AL-P were very different from those onto α-Al2O3 but quite similar to those onto HA. It is speculated that BSA is adsorbed onto AL-P and HA by interionic interactions, while it is adsorbed onto α-Al2O3 by electrostatic attraction. The results suggest that the specific adsorption of albumin onto implant materials might play a role in the expression of the bone-bonding abilities of the materials

Targeting the IGF-axis potentiates immunotherapy for pancreatic ductal adenocarcinoma liver metastases by altering the immunosuppressive microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy, resistant to chemotherapy and associated with high incidence of liver metastases and poor prognosis. Using murine models of aggressive PDAC, we show here that in mice bearing hepatic metastases, treatment with the IGF-Trap, an inhibitor of type I insulin-like growth factor receptor (IGF-IR) signaling, profoundly altered the local, immunosuppressive tumor microenvironment in the liver, curtailing the recruitment of myeloid-derived suppressor cells, reversing innate immune cell polarization and inhibiting metastatic expansion. Significantly, we found that immunotherapy with anti-PD-1 antibodies also reduced the growth of experimental PDAC liver metastases, and this effect was enhanced when combined with IGF-Trap treatment, resulting in further potentiation of a T-cell response. Our results show that a combinatorial immunotherapy based on dual targeting of the prometastatic immune microenvironment of the liver via IGF blockade, on one hand, and reversing T-cell exhaustion on the other, can provide a significant therapeutic benefit in the management of PDAC metastases.Fil: Hashimoto, Masakazu. McGill University; CanadáFil: Konda, John David. McGill University; CanadáFil: Perrino, Stephanie. McGill University; CanadáFil: Fernández, María Celia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; Argentina. McGill University; CanadáFil: Lowy, Andrew M.. Moores Cancer Centre; Estados UnidosFil: Brodt, Pnina. McGill University; Canad

Assessment of skin inflammation using near-infrared Raman spectroscopy combined with artificial intelligence analysis in an animal model

Raman spectroscopy is a powerful method for estimating the molecular structure of a target that can be adapted for biomedical analysis given its non-destructive nature. Inflammatory skin diseases impair the skin’s barrier function and interfere with the patient’s quality of life. There are limited methods for non-invasive and objective assessment of skin inflammation. We examined whether Raman spectroscopy can be used to predict skin inflammation with high sensitivity and specificity when combined with artificial intelligence (AI) analysis. Inflammation was chemically induced in mouse ears, and Raman spectra induced by a 785 nm laser were recorded. A principal component (PC) analysis of the Raman spectra was performed to extract PCs with the highest percentage of variance and to estimate the statistical score. The accuracy in predicting inflammation based on the Raman spectra with or without AI analysis was assessed using receiver operating characteristic (ROC) curves. We observed some typical changes in the Raman spectra upon skin inflammation, which may have resulted from vasodilation and interstitial oedema. The estimated statistical scores based on spectral changes correlated with the histopathological changes in the skin. The ROC curve based on PC2, which appeared to include some spectral features, revealed a maximum accuracy rate of 80.0% with an area under the curve (AUC) of 0.864. The AI analysis improved the accuracy rate to 93.1% with an AUC of 0.972. The current findings demonstrate that the combination of Raman spectroscopy with near-infrared excitation and AI analysis can provide highly accurate information on the pathology of skin inflammation

Profiling the mouse brain endothelial transcriptome in health and disease models reveals a core blood-brain barrier dysfunction module.

Blood vessels in the CNS form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells with peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizure, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders

Luminosity Distribution of Gamma-Ray Burst Host Galaxies at redshift z=1 in Cosmological Smoothed Particle Hydrodynamic Simulations: Implications for the Metallicity Dependence of GRBs

We study the relationship between the metallicity of gamma-ray burst (GRB) progenitors and the probability distribution function (PDF) of GRB host galaxies as a function of luminosity using cosmological hydrodynamic simulations of galaxy formation. We impose a maximum limit to the gas metallicity in which GRBs can occur, and examine how the predicted luminosity PDF of GRB host galaxies changes in the simulation. We perform the Kolmogorov-Smirnov test, and show that the result from our simulation agrees with the observed luminosity PDF of core-collapse supernovae (SNe) host galaxies when we assume that the core-collapse SNe trace star formation. When we assume that GRBs occur only in a low-metallicity environment with Z\lesssim 0.1 \Zsun, GRBs occur in lower luminosity galaxies, and the simulated luminosity PDF becomes quantitatively consistent with the observed luminosity PDF. The observational bias against the host galaxies of optically dark GRBs owing to dust extinction may be another reason for the lower luminosities of GRB host galaxies, but the observed luminosity PDF of GRB host galaxies cannot be reproduced solely by the dust bias in our simulation.Comment: 11 pages, 14 figures, minor revisions, one added figure, accepted for publication in Ap