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Introduction: Exchange nailing (EN) or augmentation plating (AP) has been employed to treat nonunions after intramedullary nailing for femoral shaft fractures. Although instability is a factor in hypertrophic nonunion, mechanical evaluations have been limited because the contribution of the callus to fracture site stability varies with healing. Our previous study illustrated the potential for evaluation using a finite element analysis (FEA) that incorporates callus material properties. This study aimed to mechanically evaluate revision surgery for nonunions using FEA., Materials and Methods: A quantitative computed tomography-based FEA was performed on virtual revision models of a patient with suspected nonunion after intramedullary nailing. In addition to the initial nailing model (IN) with an 11-mm diameter (D) and 360-mm length (L), four EN models with D12mm (EN1), D13mm (EN2), D12mm-L400mm (EN3), and D13mm-L400mm (EN4) nails and three AP models with 5- (AP1), 6- (AP2), and 7-hole (AP3) plates were created. As with bone, callus was assigned inhomogeneous material properties derived from density based on an empirical formula. The hip joint reaction force and muscle forces at maximum load during the gait cycle were applied. The volume ratio of the callus at the fracture site with a tensile failure risk of ≥1 (tensile failure ratio) and bone fragment movement were evaluated., Results: The tensile failure ratio was 11.6 % (IN), 10.1 % (EN1), 6.3 % (EN2), 10.9 % (EN3), 6.2 % (EN4), 6.4 % (AP1), 7.2 % (AP2), and 7.7 % (AP3), respectively. The bone fragment movement showed an opening on the lateral side with the initial intramedullary nailing. However, both revision surgeries reduced the opening, leading to compression except in the EN1 model. The proximal bone fragments were internally rotated relative to the distal fragments, and the rotational instability was more suppressed in models with lower tensile failure ratio., Conclusions: For EN, the increase in diameter, not length, is important to suppress instability. AP reduces instability, comparable to a 2 mm increase in nail diameter, and screw fixation closer to the fracture site reduces instability. This study suggest that AP is mechanically equivalent to EN and could be an option for revision surgery for femoral shaft nonunions., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

4β-Hydroxycholesterol (4β-OHC) is formed by Cytochrome P450 (CYP)3A and has drawn attention as an endogenous phenotyping probe for CYP3A activity. However, 4β-OHC is also increased by cholesterol autooxidation occurring in vitro due to dysregulated storage and in vivo by oxidative stress or inflammation, independent of CYP3A activity. 4α-hydroxycholesterol (4α-OHC), a stereoisomer of 4β-OHC, is also formed via autooxidation of cholesterol, not by CYP3A, and thus may have clinical potential in reflecting the state of cholesterol autooxidation. In this study, we establish a sensitive method for simultaneous quantification of 4β-OHC and 4α-OHC in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry. Plasma samples were prepared by saponification, two-step liquid-liquid extraction, and derivatization using picolinic acid. Intense [M+H]+ signals for 4β-OHC and 4α-OHC di-picolinyl esters were monitored using electrospray ionization. The assay fulfilled the requirements of the US Food and Drug Administration guidance for bioanalytical method validation, with a lower limit of quantification of 0.5 ng/ml for both 4β-OHC and 4α-OHC. Apparent recovery rates from human plasma ranged from 88.2% to 101.5% for 4β-OHC, and 91.8% to 114.9% for 4α-OHC. Additionally, matrix effects varied between 86.2% and 117.6% for 4β-OHC and between 89.5% and 116.9% for 4α-OHC. Plasma 4β-OHC and 4α-OHC concentrations in healthy volunteers, stage 3-5 chronic kidney disease (CKD) patients, and stage 5D CKD patients as measured by the validated assay were within the calibration ranges in all samples. We propose this novel quantification method may contribute to accurate evaluation of in vivo CYP3A activity., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

CLP1 plays an essential role in the protein complex involved in mRNA 3'-end formation and polyadenylation as well as in the tRNA splicing endonuclease (TSEN) complex involved in the splicing of precursor tRNAs. NOL9 localizes in the nucleolus of cells and plays an essential role in ribosomal RNA maturation. Both CLP1 and NOL9 are RNA kinases that phosphorylate the 5' end of RNAs. From the evidence that phosphorylation of the 5' end of a siRNA is essential for its efficient RNA cleavage, it was expected that CLP1 and NOL9 would be corresponding molecules. However, there had been no direct evidence that this is the case. In this study, murine NOL9 showed no apparent RNA kinase activity in cells or even in an RNA kinase assay using recombinant murine NOL9 protein. Although siRNA efficiency was decreased in CLP1 kinase-dead (Clp1 K/K ) cells, it was not influenced by NOL9 overexpression. These findings indicate that in mouse cells it is CLP1 that mainly acts to phosphorylate the 5' end of RNAs in the siRNA pathway, with no apparent involvement of NOL9., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: S-288310, a cancer peptide vaccine composed of two HLA-A*24:02-restricted peptides derived from two oncoantigens, DEP domain-containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1), was investigated in urothelial carcinoma (UC) of the bladder., Patients and Methods: Thirty eight HLA-A*24:02-positive patients with progressive UC were enrolled in this study. In the phase I part of the study, three patients each were treated with S-288310 at 1 mg or 2 mg/peptide subcutaneously once a week to evaluate safety and tolerability. In the phase II, 32 patients were randomized to receive either 1 mg or 2 mg to evaluate the difference in cytotoxic T lymphocytes (CTL) induction and safety., Results: S-288310 was safe and well tolerated in the phase I. Of 27 patients evaluable for immune responses in the phase II, there was no difference in CTL induction rate between the 1 mg (100%) and 2 mg (80.0%) groups. Of 32 patients receiving S-288310 in the phase II, the most frequent drug-related AE was the injection site reaction that was observed in 29 patients (90.6%), but none of the patients discontinued administration due to these reactions and no dose relationship in the frequency and severity was observed. The objective response rate of the 32 patients was 6.3% and the disease control rate was 56.3%. The median overall survival (OS) rates for patients vaccinated with S-288310 after one regimen of chemotherapy, 2 regimens, or 3 or more were 14.4, 9.1 and 3.7 months, respectively, and 32.2% of patients post first-line treatment were alive at 2 years. OS of patients who showed CTL induction to both peptides was longer than that of those with CTL induction to no or one peptide., Conclusion: S-288310 was well-tolerated and effectively induced peptide-specific CTLs, which were correlated with longer survival for patients with UC of the bladder., Trial Registration Id: JapicCTI-090980., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To examine the utility and safety of photodynamic diagnosis (PDD) after oral administration of 5-aminolevulinic acid (5-ALA) (ALA-PDD) of non-muscle-invasive bladder cancer (NMIBC) using fluorescent-light (FL)-cystoscopy., Methods: The study was a single-arm, open-label, multi-center prospective study on ALA-PDD of NMIBC, with safety as the primary endpoint and efficacy as the secondary endpoint. Diagnostic potential was evaluated through comparisons with the conventional diagnostic method using a white-light (WL)-source. Clinically recommended doses were also examined. Oral administration of 5-ALA (1.0g/50mL) was performed 180-240min before FL-cystoscopy, and positive or negative results were judged using a WL-source and based on presence or absence of red fluorescence on exposure to a blue FL-source., Results: Regarding safety, the adverse drug reactions were observed as grade 1 pruritus in 1 patient (0.6%). As for efficacy, specificity and positive predictability were lower than those of a WL-source, but sensitivity was higher with a FL-source than with a WL-source. The proportion of patients with tumors detected only by FL-cystoscopy was greater than the proportion of patients with tumors detected only by conventional WL-cystoscopy. Moreover, not only sensitivity, but also the proportion of patients with tumors detected only by FL-cystoscopy, was highest among patients who received 5-ALA at ≧20mg/kg/body., Conclusions: ALA-PDD was shown to be safe and effective. Furthermore, diagnostic accuracy of PDD increased with increased dose of 5-ALA, and the recommended dose was determined as ≧20mg/kg/body in the present study., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Recently, increased plasma mid-regional pro-adrenomedullin (MR-proADM) concentrations have been demonstrated in patients with chronic renal failure. In this study, we attempted to identify significant clinical factors associated with MR-proADM concentration in stable kidney transplant recipients., Methods: Forty-seven Japanese kidney transplant recipients who underwent transplantation >180days prior to the study were analyzed. To facilitate comparability of anti-hypertensive regimens across recipients taking different drugs, we calculated the treatment intensity score of anti-hypertensive drugs in each recipient. Morning blood samples were collected and plasma MR-proADM concentrations were measured using an enzyme immunoassay., Results: Multiple regression analysis identified treatment intensity score for anti-hypertensive drugs, serum albumin, creatinine clearance and use of lipid-lowering agents as significant independent factors associated with plasma MR-proADM concentration. Adjusted coefficient of determination for this model was 0.46., Conclusion: Apart from indicating lowered renal function, plasma MR-proADM concentration may be a useful biomarker for metabolic disorders, especially hypertension and hyperlipidemia, in stable kidney transplant patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: We had previously reported that chronic hypoxia induces androgen-independent growth in the human prostate cancer cell line LNCaP. In this study, we have identified a key molecule, the Vav3 oncogene, and investigated the effects of Vav3 overexpression on cancer cell growth and malignant behavior and the possible apoptosis-inducing effect of Vav3 expression knockdown by small interfering ribonucleic acid (siRNA) in LNCaP cells under chronic hypoxia (LNCaP/CH)., Methods and Materials: Hypoxia-inducible oncogenes were identified by complementary deoxyribonucleic acid (cDNA) microarray and Ingenuity Pathway Analysis in order to investigate gene ontology and functional pathways and networks. siRNA was used to knockdown the Vav3 target gene and analyze the effects on proliferation, invasion, migration, and apoptosis of LNCaP/CH cells. Vav3 cDNA was transfected into LNCaP cells under normoxia (LNCaP/N) to establish Vav3-overexpressing clonal cell lines, whose proliferation, invasion, and migration was then examined. Immunoblot analysis was used to investigate the activation of Akt, a Vav3 downstream target molecule., Results: cDNA microarray analysis and Ingenuity Pathway Analysis identified Vav3 as a hypoxia-inducible oncogene that was highly associated with malignant behavior. Vav3 messenger RNA and protein expression in LNCaP/CH cells were higher than in LNCaP/N and LNCaP cells cultured under acute hypoxia (LNCaP/AH). The growth rate of LNCaP/CH cells was lower than that of LNCaP/N cells but higher than that of LNCaP/AH cells. LNCaP/CH cells showed higher invasion and migration than LNCaP/N and LNCaP/AH cells. Interrupting Vav3 expression strongly suppressed the proliferation, invasion, and migration of LNCaP/CH cells. Furthermore, siRNA led to apoptosis with increased caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase activation in LNCaP/CH cells. Stable Vav3 overexpression in LNCaP cells promoted cell proliferation, invasion, and migration with Akt activation., Conclusions: Our results demonstrate that Vav3 plays a crucial role in prostate cancer growth and malignant behavior, thus revealing a novel potential therapeutic target., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: Tumor hypoxia is a common feature of any cancer, including prostate cancer (CaP), and associated with tumor cell aggressiveness. Although some reports are available on acute hypoxia-response in CaP cells aggressiveness, little is known about chronic hypoxia-response. We investigated the effects of chronic hypoxia on human CaP cells., Materials and Methods: The human androgen-dependent CaP cell line LNCaP was cultured under normoxia (21% O2), acute hypoxia (1% O2), or chronic hypoxia (1% O2 for over 6 months). The cell growth, cell cycle and cell behavior of these cells were analyzed by cell count, flow cytometric analysis and in vitro cell migration and invasion assay, respectively. The expression of matrix metalloproteinases and intracellular signaling pathways were tested by real time reverse transcriptase-polymerase chain reaction and Western blotting., Results: Chronic hypoxia-conditioned LNCaP cells grew in an androgen-independent manner with acceleration of G1 to S phase cell cycle progression. Chronic hypoxia, but not acute hypoxia, accelerated cell migration and invasion. The expressions of matrix metalloproteinase-7, -9, -14, and -15 were significantly up-regulated in LNCaP cells under chronic hypoxia, but not under acute hypoxia. In addition, PI3K/Akt, JAK/STAT, and HIF-1 pathways were activated in chronic hypoxia-conditioned LNCaP cells., Conclusions: These results suggested that chronic hypoxia plays an important role in enhancement of malignant potential during androgen-independent CaP progression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4β-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4β-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4β-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.

The purpose of the present study is to investigate the role of X-linked inhibitor of apoptosis protein (XIAP) in the regulation of apoptosis induced by cisplatin in human prostate cancer cell line (LNCaP). We examined the effects of cisplatin on cell growth and apoptosis in LNCaP by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1), flow cytometric analyses, and caspase-3 activity assay. In addition, to clarify the roles of the XIAP, we established clonal cell lines that overexpressed XIAP. The effects of cisplatin on the XIAP expression in the induction of apoptosis in LNCaP were examined by RT-PCR and immunoblot analyses. Although the growth rates were reduced in a dose- and time-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in XIAP stably overexpressing cell lines. In addition, we found that cisplatin-induced apoptosis following activation of caspase-3, and that the overexpression of XIAP inhibited apoptosis by attenuating caspase-3 activity. Interestingly, treatment of LNCaP cells with 10 and 100 muM cisplatin for 48 h significantly decreased the expression of XIAP at both the protein and mRNA levels in a dose-dependent manner. Furthermore, 10-muM cisplatin treatment of LNCaP decreased XIAP mRNA and protein in a time-dependent manner. These results suggest that cisplatin induces apoptosis by the inhibition of XIAP expression, and that XIAP plays an important role in the regulation of cisplatin-induced apoptosis in LNCaP cells. The ability of cisplatin to down-regulate XIAP may be an important mechanism in chemosensitivity.