Utilization of the pericapsular nerve group block in preoperative rehabilitation of patients with femoral neck fractures: a case series.

Background: Elderly patients with femoral neck fractures, particularly those with severe comorbidities or living in regions with limited medical resources, may experience delays in surgical treatment. Although the benefits of preoperative rehabilitation (prehabilitation) in hip arthroplasty have been reported, pain management remains a challenge. The pericapsular nerve group (PENG) block, known for its exceptional analgesic effect and motor function preservation, may be a promising intervention during prehabilitation in these patients. Case: We enrolled ten patients with Garden classification 3-4 femoral neck fractures scheduled for hip arthroplasty. After receiving a PENG block with 20 ml of 0.375% ropivacaine, all patients underwent initial prehabilitation sessions comprising 9 mobility levels, ranging from bed-sitting to walking. One patient was excluded due to experiencing high blood pressure during prehabilitation. Six of the nine remaining patients (66.7%) were successfully transferred from bed to wheelchair. Conclusions: The PENG block enhanced prehabilitation for patients with femoral neck fractures undergoing hip arthroplasty.

Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas.

To predict the prognosis of neuroblastoma patients and choose a better therapeutic protocol, we developed a cDNA microarray carrying 5340 genes obtained from primary neuroblastomas and examined 136 tumor samples. We made a probabilistic output statistical classifier that provided a high accuracy in prognosis prediction (89% at 5 years) and a highly reliable method to validate it. Kaplan-Meier analysis indicated that the patients in an intermediate group defined by existing markers are divided by microarray into two further groups with 5 year survivals for 36% and 89% of patients (p < 10(-4)), i.e., with unfavorably and favorably predicted neuroblastomas, respectively. According to these results, we developed a gene subset chip for a clinical tool, for which our classifier exhibited 88% prediction accuracy.

Cytotoxic T lymphocyte-based control of simian immunodeficiency virus replication in a preclinical AIDS vaccine trial.

Recently, encouraging AIDS vaccine trials in macaques have implicated cytotoxic T lymphocytes (CTLs) in the control of the simian human immunodeficiency virus SHIV89.6P that induces acute CD4(+) T cell depletion. However, none of these vaccine regimens have been successful in the containment of replication of the pathogenic simian immunodeficiency viruses (SIVs) that induce chronic disease progression. Indeed, it has remained unclear if vaccine-induced CTL can control SIV replication. Here, we show evidence suggesting that vaccine-induced CTLs control SIVmac239 replication in rhesus macaques. Eight macaques vaccinated with DNA-prime/Gag-expressing Sendai virus vector boost were challenged intravenously with SIVmac239. Five of the vaccinees controlled viral replication and had undetectable plasma viremia after 5 wk of infection. CTLs from all of these five macaques rapidly selected for escape mutations in Gag, indicating that vaccine-induced CTLs successfully contained replication of the challenge virus. Interestingly, analysis of the escape variant selected in three vaccinees that share a major histocompatibility complex class I haplotype revealed that the escape variant virus was at a replicative disadvantage compared with SIVmac239. These findings suggested that the vaccine-induced CTLs had "crippled" the challenge virus. Our results indicate that vaccine induction of highly effective CTLs can result in the containment of replication of a highly pathogenic immunodeficiency virus.

[The use of total enteral formula with enriched nutrient in home Low Calorie Diet(LCD)therapy based on regular meal].

We made a low calorie diet(LCD)menu which added two commercial supporting nutritional supplements to a meal. Because a conventional formula food is very expensive, the patient was not able to afford it at home. Those supplements are a total enteral formula with enriched nutrient(ACURE EN800)and vitamin-mineral rich drink(V CRESC). The contents of vitamin and mineral in this menu satisfied the dietary reference intakes, though protein was a little low. However, we could keep the price low compared to the formula food. The patient was able to switch over to home LCD therapy with the menu.

AcrS/EnvR represses expression of the acrAB multidrug efflux genes in Escherichia coli.

The acrS regulatory gene is located upstream of the acrEF multidrug efflux system genes. However, the roles of AcrS in regulation of drug efflux pumps have not been clearly understood. Here we show that AcrS represses other multidrug efflux genes, acrAB, which encode a major efflux system in Escherichia coli.

Involvement of mast cells in the regulation of matrix metalloproteinase-9 and tissue inhibitor of metalloproteases-1 in 12-O-tetradecanoylphorbolacetate-induced inflammation in mice.

In this study, we investigated the involvement of mast cells in the regulation of matrix metalloproteinase-9 (MMP-9) in 12-O-tetradecanoylphorbolacetate (TPA)-induced inflammation, using mast cell-deficient (W/W(v)) mice and control (+/+) mice. Topical application of TPA to the ears induced acute inflammation, accompanied by mast cell degranulation in +/+ mice, which peaked at 6-12 h. There was no significant difference in ear thickness between the groups until 12 h, but the swelling was greater in W/W(v) mice than +/+ mice at 24-36 h. Western blot analysis revealed that TPA-induced marked increases in levels of proMMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1), which existed as complexes with proMMP-9. The amount of proMMP-9-TIMP-1 complex was markedly smaller in +/+ mice than W/W(v) mice at 6 and 24 h, but had almost returned to control levels in both groups at 48 h. The free form of proMMP-9 was also slightly less abundant in +/+ mice than W/W(v) mice at 6, 24, and 48 h. Gelatin zymographic analysis revealed that levels of the active species of MMP-9 (approximately 74 and 83 kD), as well as free form of proMMP-9, increased time-dependently after the application of TPA and peaked at 24 h in +/+ mice. The 74-kD band was detected only in +/+ mice at 6 h. Our results therefore suggested that during inflammation degranulation of mast cells results in a reduction of the proMMP-9-TIMP-1 complex levels, together with a fall in the amount of free proMMP-9.

Expression profiling and differential screening between hepatoblastomas and the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas.

Hepatoblastoma is one of the most common malignant liver tumors in young children. Recent evidences have suggested that the abnormalities in Wnt signaling pathway, as seen in frequent mutation of the beta-catenin gene, may play a role in the genesis of hepatoblastoma. However, the precise mechanism to cause the tumor has been elusive. To identify novel hepatoblastoma-related genes for unveiling the molecular mechanism of the tumorigenesis, a large-scale cloning of cDNAs and differential screening of their expression between hepatoblastomas and the corresponding normal livers were performed. We constructed four full-length-enriched cDNA libraries using an oligo-capping method from the primary tissues which included two hepatoblastomas with high levels of alpha-fetoprotein (AFP), a hepatoblastoma without production of AFP, and a normal liver tissue corresponded to the tumor. Among the 10,431 cDNAs randomly picked up and successfully sequenced, 847 (8.1%) were the genes with unknown function. Of interest, the expression profile among the two subsets of hepatoblastoma and a normal liver was extremely different. A semiquantitative RT-PCR analysis showed that 86 out of 1188 genes tested were differentially expressed between hepatoblastomas and the corresponding normal livers, but that only 11 of those were expressed at high levels in the tumors. Notably, PLK1 oncogene was expressed at very high levels in hepatoblastomas as compared to the normal infant's livers. Quantitative real-time RT-PCR analysis for the PLK1 mRNA levels in 74 primary hepatoblastomas and 29 corresponding nontumorous livers indicated that the patients with hepatoblastoma with high expression of PLK1 represented significantly poorer outcome than those with its low expression (5-year survival rate: 55.9 vs 87.0%, respectively, p=0.042), suggesting that the level of PLK1 expression is a novel marker to predict the prognosis of hepatoblastoma. Thus, the differentially expressed genes we have identified may become a useful tool to develop new diagnostic as well as therapeutic strategies of hepatoblastoma.

A review of DNA microarray analysis of human neuroblastomas.

Neuroblastoma (NBL) is an enigmatic tumor with heterogeneous clinical behaviors including maturation, regression, and aggressive growth. Despite recent progress in therapeutic strategies against advanced NBL, long-term outcomes still remain very poor. The prediction of cancer prognosis is one of the most urgent demands to initiate the suitable treatment of NBL. Recent papers have demonstrated that cancers can be diagnosed on the basis of gene expression profiling. We have been proceeded NBL cDNA project to collect a large number of genes expressed in NBLs, to identify the genes differentially expressed between favorable and unfavorable NBLs, and to make an NBL-proper cDNA chip for large-scale analysis of NBL tumors. Computational analysis of gene expression data in NBLs identified many prognosis-related genes and provided a classifier to predict the patient prognosis with high efficiency. Conversion of these findings into better diagnosis and treatment is now underway. Thus, molecular profiling of NBL has become a feasible tool for clinical applications.

Membrane topology of ABC-type macrolide antibiotic exporter MacB in Escherichia coli.

MacB is an ABC-type membrane protein that exports only macrolide compounds containing 14- and 15-membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site-specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N-terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.

Recombinant Sendai viruses expressing different levels of a foreign reporter gene.

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative strand RNA genome. The recovery of infectious virus from cDNA and generation of recombinant SeV carrying a foreign gene at the promoter proximal position has been demonstrated. In this study, we constructed a series of recombinant SeVs carrying a reporter human secreted alkaline phosphatase (SEAP) gene at each viral gene junction or the 5' distal end in order to measure the expression level of the foreign gene. We demonstrated that there was a gradient in the reporter gene expression level that depended on location, due to the polarity of transcription. In contrast, the growth and final titers of these recombinant viruses showed an opposite gradient to the foreign gene expression level. This suggests the potential for matching therapeutic gene expression level to individual therapy programs by changing the position of the foreign gene when SeVs are used as vectors for human gene therapy.

An improved method for recovery of F-defective Sendai virus expressing foreign genes from cloned cDNA.

An improved system is described to recover non-transmissible Sendai virus that lack the envelope fusion (F) gene from cloned cDNA. The system (1) used plasmids that expressed the F and the HN viral envelope proteins, as well as the plasmids that expressed the viral NP, P, and L proteins as helper plasmids for recovery, and (2) overlaid packaging cells that expressed the F protein. With this improved system, we have succeeded in recovery of F-defective Sendai virus expressing two foreign proteins, and expression vectors that do not contain the EGFP reporter gene. This system may provide the basis for constructing recombinant F-defective Sendai virus for preventing and treating human diseases in the form of vaccines and vectors for gene therapy.

[An example of self-evaluation of a sense of achievement by students in 6-year pharmacy school with the model core curriculum of pharmaceutical education].

In March 2012, the first students, finishing the newly introduced 6-year-course of pharmaceutical education, have graduated and gone out into the world. At this point, the Ministry of Education, Culture, Sports, Science and Technology (MEXT) is going to revise the model core curriculum of pharmaceutical education to be more suited for educating students to achieve their goal of becoming the clinical pharmacist standard defined by the revised School Education Act. Here we report the self-evaluation study based on the survey using questionnaire about a sense of achievement with Visual Analog Scales, regarding the fundamental quality as a pharmacist standard proposed by the Professional Activities Committee in the MEXT. The sample size of survey was about 600 of students studying in the Faculty of Pharmaceutical Sciences in Josai International University (JIU) and the survey was carried out during the period of March-April in 2012. The study suggested that the majority of graduates were satisfied with the new education system and marked as a well-balanced quality to be a pharmacist standard, after completing the 6-year pharmaceutical education based on "the model core-curriculum". It would be worthwhile to perform this kind of survey continuously to monitor the student's self-evaluation of a sense of achievement to verify the effectiveness of 6-year-course pharmaceutical education based on the newly establishing core curriculum in Japan.

Growth phase-dependent expression of drug exporters in Escherichia coli and its contribution to drug tolerance.

Drug exporters contribute to the intrinsic drug resistance in many organisms. Although there are at least 20 exporter genes in Escherichia coli, most of them apparently do not confer drug resistance in complex laboratory media except for the AcrAB, EmrE, and MdfA efflux systems. In this study, we comprehensively investigated the growth phase-dependent expression of drug exporter genes. The expression of acrAB, emrAB, emrD, emrE, emrKY, mdfA, and ydgFE is stable at moderate levels during any growth phase, whereas mdtEF promoter activity greatly increased with cell growth and reached the maximum level at the late stationary phase. The growth phase-dependent increase in mdtEF expression was also observed on quantitative reverse transcription-PCR analysis. As expected from the transporter expression, the stationary-phase cells actually showed MdtEF-dependent tolerance to drugs and toxic dyes. Growth phase-dependent elevation of mdtEF expression was found to be mediated by the stationary-phase sigma factor rpoS and the RpoS-dependent signaling pathway, Hfq, GadY, and GadX. The induction level was decreased by tnaAB deletion, suggesting that indole sensing stimulates this process.

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner.

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca(2+). Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca(2+)-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca(2+) also stimulated S1P release from ATX platelets without ATP, whereas the Ca(2+)-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca(2+).

N-acetyl-d-glucosamine induces the expression of multidrug exporter genes, mdtEF, via catabolite activation in Escherichia coli.

The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-d-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and d-glucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.

Indole induces the expression of multidrug exporter genes in Escherichia coli.

Our comprehensive expression cloning studies previously revealed that 20 intrinsic xenobiotic exporter systems are encoded in the Escherichia coli chromosome, but most of them are not expressed under normal conditions. In this study, we investigated the compounds that induce the expression of these xenobiotic exporter genes, and found that indole induces a variety of xenobiotic exporter genes including acrD, acrE, cusB, emrK, mdtA, mdtE and yceL. Indole treatment of E. coli cells confers rhodamine 6G and SDS resistance through the induction of mdtEF and acrD gene expression respectively. The induction of mdtE by indole is independent of the EvgSA two-component signal transduction system that regulates the mdtE gene, but mediated by GadX. On the other hand, the induction of acrD and mdtA was mediated by BaeSR and CpxAR, two-component systems. Interestingly, CpxAR system-mediated induction required intrinsic baeSR genes, whereas BaeSR-mediated induction was observed in the cpxAR gene-deletion mutant. BaeR and CpxR directly bound to different sequences of the acrD and mdtA promoter regions. These observations indicate that BaeR is a primary regulator, and CpxR enhances the effect of BaeR.

Comprehensive studies of drug resistance mediated by overexpression of response regulators of two-component signal transduction systems in Escherichia coli.

In Escherichia coli, there are 32 open reading frames (ORFs) that are assumed to be response regulator genes of two-component signal transduction systems on the basis of sequence similarities. We cloned all of these 32 ORFs into a multicopy expression vector and investigated whether or not they confer drug resistance via control of drug resistance determinants. Fifteen of these ORFs, i.e., baeR, citB, cpxR, evgA, fimZ, kdpE, narL, narP, ompR, rcsB, rstA, torR, yedW, yehT, and dcuR, conferred increased single- or multidrug resistance. Two-thirds of them conferred deoxycholate resistance. Five of them, i.e., evgA, baeR, ompR, cpxR, and rcsB, modulated the expression of several drug exporter genes. The drug resistance mediated by evgA, baeR, and cpxR could be assigned to drug exporters by using drug exporter gene knockout strains.

The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC.

Overproduction of the response regulator BaeR confers resistance to novobiocin and bile salts in a DeltaacrAB mutant by stimulating drug exporter gene expression. The mdtABC (multidrug transporter ABC, formerly known as yegMNO) genes, which encode a resistance-nodulation-cell division (RND) drug efflux system, are responsible for resistance. The MdtABC system comprises the transmembrane MdtB/MdtC heteromultimer and MdtA membrane fusion protein. MdtAC also confers bile salt, but not novobiocin, resistance. This indicates that the evolution from an MdtC homomultimer to an MdtBC heteromultimer contributed to extend the drug resistance spectrum. A BLAST search suggested that such a heteromultimer-type RND exporter constitutes a unique family among gram-negative organisms.

Effects of efflux transporter genes on susceptibility of Escherichia coli to tigecycline (GAR-936).

The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 microM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters.