Early Rehabilitation with Weight-bearing Standing-shaking-board Exercise in Combination with Electrical Muscle Stimulation after Anterior Cruciate Ligament Reconstruction

The objective of early rehabilitation after anterior cruciate ligament (ACL) reconstruction is to increase the muscle strength of the lower extremities. Closed kinetic chain (CKC) exercise induces co-contraction of the agonist and antagonist muscles. The purpose of this study was to compare the postoperative muscle strength/mass of subjects who performed our new CKC exercise (new rehabilitation group:group N) from week 4, and subjects who received traditional rehabilitation alone (traditional rehabilitation group:group T). The subjects stood on the device and maintained balance. Then, low-frequency stimulation waves were applied to 2 points each in the anterior and posterior region of the injured thigh 3 times a week for 3 months. Measurement of muscle strength was performed 4 times (before the start, and then once a month). Muscle mass was evaluated in CT images of the extensor and flexor muscles of 10 knees (10 subjects) in each group. The injured legs of group N showed significant improvement after one month compared to group T. The cross-sectional area of the extensor muscles of the injured legs tended to a show a greater increase at 3 months in group N. This rehabilitation method makes it possible to contract fast-twitch muscles, which may be a useful for improving extensor muscle strength after ACL reconstruction

Prenatal diagnosis of Jacobsen syndrome with cystic hygroma

Jacobsen syndrome (JBS) is a rare, contiguous genetic deletion syndrome caused by partial deletion of chromosome 11q. It is difficult to diagnose JBS by ultrasonography during the prenatal period because it has atypical clinical findings. Although nuchal thickening is associated with JBS, cystic hygroma has not been previously reported in conjunction with this syndrome. We describe the first prenatal diagnosis of JBS with cystic hygroma, which was clinically diagnosed at 13 weeks gestation in a Japanese woman, although it had not been detectable at the 11th week of gestation. In addition, a single ventricle fetal heart defect was suspected. Amniocentesis was performed, and G-banded karyotype evaluation revealed 46,XX,del(11)(q23), which is consistent with JBS. Cystic hygroma and congenital heart malformation have been reported to be associated with chromosomal anomalies. The present case indicates that a fetal cystic hygroma without increased nuchal thickness might be associated with JBS

2000-2001年の太平洋西部海域におけるプラスチック微小片の分布

Sampling was conducted at 31 sites in the western Pacific Ocean from 2000 to 2001 with the aim of collecting plastic fragments with a neuston net (mesh size: 1.00 mm × 1.64 mm). Small plastic fragments including microplastics (small fragments in the size range of 1.1–41.8 mm) were collected at multiple survey sites. Waters with high densities of small fragments were observed between 20°N and 30°N to the south of Japan and between 20°S and 30°S to the northeast of New Zealand (maxima of 6.63 × 102 and 2.04 × 102 pieces/ha, respectively). These waters are located to the west of the Ekman convergence zones related to trade winds in the subtropical gyres of the North and South Pacific Oceans. Nearly no small plastics were observed in the tropical circulation of the western Pacific Ocean.2000年から2001年にかけて太平洋西部海域の31地点でニューストンネット（目合1.00mm×1.64mm）によるプラスチック微小片の採集を行った。マイクロプラスチック(5mm以下)を含むプラスチック微小片は，日本の南の北緯20—30度の海域とニュージーランド北東の南緯20—30度の海域で高密度に採集され（各々最高で6.63×102と2.04×102個/ha），これらの海域は北太平洋と南太平洋における亜熱帯循環内の漂流物収束帯の西端に位置する。赤道付近の熱帯循環内の採集点ではほとんど採集されなかった

Efficient viral delivery of Cas9 into human safe harbor

Gene editing using CRISPR/Cas9 is a promising method to cure many human genetic diseases. We have developed an efficient system to deliver Cas9 into the adeno-associated virus integration site 1 (AAVS1) locus, known as a safe harbor, using lentivirus and AAV viral vectors, as a step toward future in vivo transduction. First, we introduced Cas9v1 (derived from Streptococcus pyogenes) at random into the genome using a lentiviral vector. Cas9v1 activity was used when the N-terminal 1.9 kb, and C-terminal 2.3 kb fragments of another Cas9v2 (human codon-optimized) were employed sequentially with specific single-guide RNAs (sgRNAs) and homology donors carried by AAV vectors into the AAVS1 locus. Then, Cas9v1 was removed from the genome by another AAV vector containing sgRNA targeting the long terminal repeat of the lentivirus vector. The reconstituted Cas9v2 in the AAVS1 locus was functional and gene editing was efficient

SAM domain-containing N-terminal region of SAMHD1 plays a crucial role in its stabilization and restriction of HIV-1 infection

SAMHD1 restricts human immunodeficiency virus type 1 (HIV-1) infection in a cell-type specific manner. Other than primary monocyte derived cells and resting CD4+ T cells, the SAMHD1-mediated HIV-1 block was reported only in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 and U937 monocyte cell lines. We previously reported that SAMHD1 restricted HIV-1 infection in TE671 rhabdomyosarcoma cells in addition to these cell lines. In this study, we compared the amounts of the full-length SAMHD1 and its deletion mutants, SAM domain containing N-terminal fragment (residues 1-119, SAMHD1n) and HD domain containing C-terminal fragment (120-626, SAMHD1c) in U937, TE671, and HeLa cells. The results showed that the full-length SAMHD1 and SAMHD1n proteins were significantly more abundant than the SAMHD1c protein in TE671 and differentiated U937 cells. The proteasome inhibitor MG132 increased the amount of the SAMHD1c and the SAMHD1c-fused GFP proteins. In contrast, the fusion of the SAMHD1n to the APOBEC3G protein inhibited Vif-induced proteasomal degradation in TE671 and in differentiated U937 cells. These results indicated that the SAMHD1 C-terminal HD domain-containing region leads the SAMHD1 to proteasomal degradation, and the SAMHD1 N-terminal SAM domain-containing region stabilizes the protein. Our study showed that the SAMHD1 protein expression is post-translationally regulated and the significance of SAM and HD domains for the full-length SAMHD1 protein stability. Further, we suggest that the SAM domain-containing N-terminal region participate in the cell-type specific restrictive function of SAMHD1 against HIV-1 infection, by protein stabilization

IDO1, FAT10, IFI6, and GILT Are Involved in the Antiretroviral Activity of γ-Interferon and IDO1 Restricts Retrovirus Infection by Autophagy Enhancement

Gamma-interferon (γ-IFN) significantly inhibits infection by replication-defective viral vectors derived from the human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) but the underlying mechanism remains unclear. Previously we reported that knockdown of γ-IFN-inducible lysosomal thiolreductase (GILT) abrogates the antiviral activity of γ-IFN in TE671 cells but not in HeLa cells, suggesting that other γ-IFN-inducible host factors are involved in its antiviral activity in HeLa cells. We identified cellular factors, the expression of which are induced by γ-IFN in HeLa cells, using a microarray, and analyzed the effects of 11 γ-IFN-induced factors on retroviral vector infection. Our results showed that the exogenous expression of FAT10, IFI6, or IDO1 significantly inhibits both HIV-1- and MLV-based vector infections. The antiviral activity of γ-IFN was decreased in HeLa cells, in which the function of IDO1, IFI6, FAT10, and GILT were simultaneously inhibited. IDO1 is an enzyme that metabolizes an essential amino acid, tryptophan. However, IDO1 did not restrict retroviral vector infection in Atg3-silencing HeLa cells, in which autophagy did not occur. This study found that IDO1, IFI6, FAT10, and GILT are involved in the antiviral activity of γ-IFN, and IDO1 inhibits retroviral infection by inducing autophagy

Retrovirus Entry by Endocytosis and Cathepsin Proteases

Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines

Rab3a, a small GTP-binding protein, is required for the stabilization of the murine leukaemia virus Gag protein

We recently identified a CD63-interacting protein to understand the role of CD63 in virion production of the human immunodeficiency virus type 1, and we have found that Rab3a forms a complex with CD63. In this study, we analysed the effect of Rab3a on virion production of the murine leukaemia virus (MLV), which is another member of the retrovirus family. We found that Rab3a silencing induced lysosomal degradation of the MLV Gag protein, and recovery of the Rab3a expression restored the level of the Gag protein through a complex formation of MLV Gag and Rab3a, indicating that Rab3a is required for MLV Gag protein expression. In contrast, CD63 silencing decreased the infectivity of released virions but had no effect on virion production, indicating that CD63 facilitates the infectivity of released MLV particles. Although Rab3a induced CD63 degradation in uninfected cells, the complex of MLV Gag and Rab3a suppressed the Rab3a-mediated CD63 degradation in MLV-infected cells. Finally, we found that the MLV Gag protein interacts with Rab3a to stabilize its own protein and CD63 that facilitates the infectivity of released MLV particles. Considering the involvement of Rab3a in lysosome trafficking to the plasma membrane, it may also induce cell surface transport of the MLV Gag protein

Enterokinase and IAV Infection

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines