Quadriceps muscle activity during walking with a knee ankle foot orthosis is associated with improved gait ability in acute hemiplegic stroke patients with severe gait disturbance.

Introduction: A knee-ankle-foot orthosis (KAFO) prevents knee buckling during walking and enables gait training for acute hemiplegic stroke patients with severe gait disturbances. Although the goal of gait training with a KAFO is to improve gait ability, that is, to acquire walking with an ankle-foot orthosis (AFO), it is not clear how gait training with a KAFO contributes to improving gait ability. Therefore, this study aimed to investigate the relationship between muscle activities during walking with a KAFO and the improvement of gait ability in hemiplegic stroke patients with severe gait disturbance. Methods: A prospective cohort study was conducted. Fifty acute hemiplegic stroke patients who could not walk with an AFO participated. Muscle activities of the paretic rectus femoris, biceps femoris, tibialis anterior, and soleus were assessed with surface electromyogram during walking with a KAFO. Electromyograms were assessed at the beginning of gait training and at the time the Ambulation Independence Measure score improved by 3 or higher, or discharge. Results: Even in patients with complete hemiplegia, paretic rectus femoris, biceps femoris, and soleus showed periodic muscle activity during walking with a KAFO. Twenty-three patients improved to an Ambulation Independence Measure score of 3 or higher and were able to walk with an AFO (good recovery group). At the beginning of gait training, paretic rectus femoris muscle activity during the first double-limb support phase was significantly higher in the good recovery group than in the poor recovery group. The rectus femoris muscle activity significantly increased from before to after acute rehabilitation, which consisted mainly of gait training with a KAFO. Discussion: For acute hemiplegic stroke patients with severe disturbance, the induction and enhancement of paretic quadriceps muscle activity during walking with a KAFO play an important role in acquiring walking with an AFO.

Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems.

Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.

Characterization of LuxI and LuxR Protein Homologs of N-Acylhomoserine Lactone-Dependent Quorum Sensing System in Pseudoalteromonas sp. 520P1.

Pseudoalteromonas sp. 520P1 (hereafter referred to as strain 520P1) produces N-acylhomoserine lactones (AHLs), which serve as signaling molecules in Gram-negative bacterial quorum sensing. In a previous genomic analysis of the 5.25-Mb genome of strain 520P1, we detected the presence of at least one homolog of the AHL synthase gene (luxI) and five homologs of the transcriptional regulator protein gene (luxR). The LuxI homolog of strain 520P1 (PalI) contained the conserved amino acid motifs shared by all the LuxI family proteins of the different species examined here. The palI gene expressed in Escherichia coli produced two types of AHLs. In the thin-layer chromatography analysis, these AHLs showed identical mobility to the AHLs produced by strain 520P1. The five LuxR homologs of strain 520P1 (PalR1-PalR5) shared only 17-34% amino acid sequence identity, although higher identities were observed in the C-terminal DNA-binding domain. Among the five PalRs, only PalR5 displayed close homology with LuxR family proteins from other Pseudoalteromonas strains. Notably, the palR3 and palI genes were located close together and only 1021 bases apart in the genome. No cognate luxI homolog associated with the four other palR genes was detected. These characteristics of PalI and the PalRs suggest that AHL autoinducers generated by the PalI enzyme might regulate cellular metabolism in cooperation with five transcriptional regulator PalRs, each of which is presumed to play a distinctive role in bacterial signaling.

Potentiation of cell invasion and matrix metalloproteinase production by alpha3beta1 integrin-mediated adhesion of gastric carcinoma cells to laminin-5.

We previously reported that the adhesion of gastric carcinoma cells to the peritoneum mediated by the alpha3beta1 integrin-laminin interaction is a key step in the initial process of peritoneal metastatic dissemination. Carcinoma cells subsequently invade through the intercellular gaps of mesothelial linings. In this study, we examined the role of the interaction of carcinoma cells with laminin-5, which is a major component of submesothelial basement membranes and serves as a high-affinity ligand for alpha3beta1 integrin, in carcinoma cell invasion. Human gastric carcinoma cell lines (MKN1, GT3TKB, and NUGC-4) adhered in an alpha3beta1 integrin-dependent manner to the extracellular matrix deposited by peritoneal mesothelial cells. An in vitro invasion assay using the Boyden chamber system revealed that MKN1 cell migration through the membranes increased when the membranes were coated with matrices produced by mesothelial cells or with laminin-5-containing Matrigel as compared to Matrigel alone. The cell migration promoted by laminin-5-containing Matrigel was inhibited by the presence of anti-alpha3 integrin antibody. When MKN1 cells were cultured in a laminin-5-coated plate, these cells were promoted to produce matrix metalloproteinase (MMP)-9, as assessed by gelatin zymography, enzyme-linked immunosorbent assay, and reverse transcription-polymerase chain reaction. These results suggest that the production of MMP-9 by MKN1 cells was potentiated by the alpha3beta1 integrin-laminin-5 interaction, which facilitated their invasion via degradation of the matrix.

Adhesion of gastric carcinoma cells to peritoneum mediated by alpha3beta1 integrin (VLA-3).

The interaction between gastric carcinoma cells and the peritoneal lining is a key step in peritoneal dissemination. In this study, we examined the roles of the beta1 family of integrin receptors in the adhesion of such cells to the peritoneum. The adhesion of several gastric carcinoma cell lines to peritonea excised from mice was inhibited most by an anti-alpha3 integrin antibody and to a lesser extent by an anti-alpha2 integrin antibody. In the peritoneal implantation of NUGC-4 human gastric carcinoma cells in athymic mice, treatment of the cells with anti-alpha2 or anti-alpha3 integrin antibody reduced the number of disseminated nodules; suppression by the anti-alpha3 integrin antibody was stronger than that by the anti-alpha2 integrin antibody. The cDNAs to human alpha2 and alpha3 integrins were introduced into K562 leukemic cells, which were positive for the integrin beta1 subunit but negative for the alpha2 or alpha3 subunit. The alpha3 integrin-transfected cells adhered to excised peritoneum and to a monolayer of peritoneal mesothelial cells more firmly than did the alpha2 integrin-transfected cells or the mock transfectant. Reverse transcription-PCR was used to analyze the expression of laminin-5 and laminin-10/11, which have been reported to serve as high-affinity ligands for alpha3beta1 integrin. mRNA for these laminin isoforms was found in mesothelial cells from the diaphragm and parietal peritoneum. These results strongly suggest that alpha3beta1 integrin plays an essential role in mediating the initial attachment of cancer cells to the peritoneum, leading to the formation of peritoneal metastasis.

Regulation of melanoma cell migration and invasion by laminin-5 and alpha3beta1 integrin (VLA-3).

We evaluated the role of soluble factors produced from epidermal cells in melanoma cell motility by using the Boyden chamber chemoinvasion system. The migration of two melanoma cell lines, A375 and Mewo, was potentiated by conditioned media of A431 epidermoid cells in a concentration-dependent manner. The enhancement of A375 melanoma cell motility induced by the conditioned medium was blocked by antibodies against either alpha3 or beta1 integrin subunit. The motility-stimulating activity was recovered in the same fraction as the alpha3 integrin-dependent adhesion-promoting activity in a high-molecular-weight (>200 kDa) fraction on Superose 12 gel chromatography, and adsorbed with an anti-laminin-5 antibody. Purified laminin-5 was capable of potentiating melanoma cell migration as measured in either the chemotaxis assay with a soluble form of laminin-5 or the haptotaxis assay with membranes coated with a mixture of laminin-5 and Matrigel. Furthermore, immobilized laminin-5 induced A375 melanoma cells to secrete matrix metalloproteinase-9 (type IV collagenase) into the culture medium. These results strongly suggest that the interaction of laminin-5 produced in the epidermis with alpha3beta1 integrin on melanoma cells is involved in cell migration, invasion, and degradation of extracellular matrix proteins.