Up-regulation of secretory leukocyte protease inhibitor in human samples might have a potential role of predicting prostate cancer recurrence and progression after surgery and hormonal therapy.

Using new castration-resistant prostate cancer (CRPC) cell lines developed from LNCaP cells as a model for CRPC, we searched for novel biomarkers by analyzing the proteins secreted in culture supernatants. The results showed that the levels of secretory leukocyte protease inhibitor (SLPI) in these cell lines were 4.7-6.7 times higher than those secreted in parental LNCaP. Patients with localized prostate cancer (PC) and who expressed SLPI had a significantly lower prostate-specific antigen (PSA) progression-free survival rate than those who did not. Multivariate analysis revealed that SLPI expression was an independent risk factor for PSA recurrence. By contrast, when immunostaining of SLPI was performed on consecutive prostate tissue samples obtained from 11 patients, both in hormone naive (HN) and castration resistant (CR) conditions, only one patient expressed SLPI in the HNPC state; however, four of the 11 patients expressed SLPI in the CRPC state. In addition, two of these four patients were resistant to enzalutamide, and there was a discrepancy between their serum PSA levels and radiographic progression of the disease. These results suggest that SLPI can be a predictor of prognosis in patients with localized PC and disease progression in CRPC patients.

High level of phosphatidylcholines/lysophosphatidylcholine ratio in urine is associated with prostate cancer.

The altered levels of phospholipids (PLs) and lysophospholipids (LPLs) in prostate cancer (CaP) and benign tissues in our previous findings prompted us to explore PLs and LPLs as potential biomarkers for CaP. Urinary lipidomics has attracted increasing attention in clinical diagnostics and prognostics for CaP. In this study, 31 prostate tissues obtained from radical prostatectomy were assessed using high-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS). Urine samples were collected after digital rectal examination (DRE), and urinary lipids were extracted using the acidified Bligh-Dyer method. The discovery set comprised 75 patients with CaP and 44 with benign prostatic hyperplasia (BPH) at Kyoto University Hospital; the validation set comprised 74 patients with CaP and 59 with BPH at Osaka University Hospital. Urinary lipidomic screening was performed using MALDI time-of-flight MS (MALDI-TOF/MS). The levels of urinary lysophosphatidylcholine (LPC) and phosphatidylcholines (PCs) were compared between the CaP and BPH groups. The (PC [34:2] + PC [34:1])/LPC (16:0) ratio was significantly higher (P < .001) in CaP tissues than in benign epithelial tissues. The urinary PCs/LPC ratio was significantly higher (P < .001) in the CaP group than in the BPH group in the discovery and validation sets.

A narrative review of urinary phospholipids: from biochemical aspect towards clinical application.

As a newly emerged discipline, lipidomic studies have focused on the comprehensive characterization and quantification of lipids in a given biological system, which has remarkably advanced in recent years owing to the rapid development of analytical techniques, especially mass spectrometry. Among diverse lipid classes, phospholipids, which have fundamental roles in the formation of cellular membranes, signaling processes, and bioenergetics have gained momentum in several fields of research. The altered composition, concentration, spatial distribution, and metabolism of phospholipids in cells, tissues, and body fluids have been elucidated in various human diseases such as cancer, inflammation, as well as cardiovascular and metabolic disorders. Among the different kinds of phospholipid sources in the human body, urine has not been extensively investigated in recent years owing to the extremely low concentrations of phospholipids and high levels of salts and other contaminants, which can interfere with precise detection. However, with profound advances and rapid expansion in analytical methods, urinary phospholipids have attracted increasing attention in current biomedical research as urine is an easily available source for the discovery of noninvasive biomarkers. In this review, we provide an overview of urinary phospholipids, including their biochemical aspects and clinical applications, aimed at promoting this field of research.

Systematic chemical screening identifies disulfiram as a repurposed drug that enhances sensitivity to cisplatin in bladder cancer: a summary of preclinical studies.

BACKGROUND: Since the standard gemcitabine and cisplatin (GC) chemotherapy for advanced bladder cancer yields limited therapeutic effect due to chemoresistance, it is a clinical challenge to enhance sensitivity to GC. METHODS: We performed high-throughput screening by using a library of known chemicals and repositionable drugs. A total of 2098 compounds were administered alone or with GC to human bladder cancer cells, and chemicals that enhanced GC effects were screened. RESULTS: Disulfiram (DSF), an anti-alcoholism drug, was identified as a candidate showing synergistic effects with cisplatin but not with gemcitabine in multiple cell lines. Co-administration of DSF with GC affected cellular localisation of a cisplatin efflux transporter ATP7A, increased DNA-platinum adducts and promoted apoptosis. Micellar DSF nanoparticles (DSF-NP) that stabilised DSF in vivo, enhanced the inhibitory effect of cisplatin in patient-derived and cell-based xenograft models without severe adverse effects. A drug susceptibility evaluation system by using cancer tissue-originated spheroid culture showed promise in identifying cases who would benefit from DSF with cisplatin. CONCLUSIONS: The present study highlighted the advantage of drug repurposing to enhance the efficacy of anticancer chemotherapy. Repurposing of DSF to a chemotherapy sensitiser may provide additional efficacy with less expense by using an available drug with a well-characterised safety profile.

Comparative evaluation of the extraction and analysis of urinary phospholipids and lysophospholipids using MALDI-TOF/MS.

Lipids, particularly phospholipids (PLs) and lysophospholipids (LPLs), are attracting increasing scientific interest for their biological functions in cells and their potential as disease biomarkers for Alzheimer's disease and several types of cancer. Urinary PLs and LPLs could be ideal clinical biomarkers, because urine can be collected easily and noninvasively. However, due to their very low concentrations in urine compared with the relatively large quantity of contaminants in this matrix, efficient extraction and sensitive detection are required for analyzing urinary PLs and LPLs. In this study, various methods for analyzing PLs and LPLs in urine were compared and optimized from a clinical perspective. An optimized lipid extraction method and a matrix for matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were established using two external ionization standards and an internal standard mix containing 13 human urinary lipids. 9-Aminoacridine (9-AA) was a useful and effective matrix for the MALDI-TOF/MS analysis of all the internal standard lipids in both positive and negative ion modes. However, it was necessary to determine the proportional lipid concentrations from the balance between the extracted lipid and the matrix. The extraction efficiency and reproducibility of the acidified Bligh and Dyer method were excellent for both positively and negatively charged lipids. Analysis of small volumes of urine was the most efficient with the 9-AA MALDI matrix at concentrations of or below 5 mM. The combined analytical procedures allowed rapid and comprehensive screening of low concentrations of PLs and LPLs in clinical samples.

Decreased expression of lysophosphatidylcholine (16:0/OH) in high resolution imaging mass spectrometry independently predicts biochemical recurrence after surgical treatment for prostate cancer.

BACKGROUND: Human prostate cancers are highly heterogeneous, indicating a need for various novel biomarkers to predict their prognosis. Lipid metabolism affects numerous cellular processes, including cell growth, proliferation, differentiation, and motility. Direct profiling of lipids in tissue using high-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) may provide molecular details that supplement tissue morphology. METHODS: Prostate tissue samples were obtained from 31 patients, with localized prostate cancer who underwent radical prostatectomy. The samples were assessed by HR-MALDI-IMS in positive mode, with the molecules identified by tandem mass spectrometry (MS/MS). The effect of identified molecules on prostate specific antigen recurrence free survival after radical prostatectomy was determined by Cox regression analysis and by the Kaplan-Meier method. RESULTS: Thirteen molecules were found to be highly expressed in prostate tissue, with five being significantly lower in cancer tissue than in benign epithelium. MS/MS showed that these molecules were [lysophosphatidylcholine (LPC)(16:0/OH)+H](+), [LPC(16:0/OH)+Na](+), [LPC(16:0/OH)+K](+), [LPC(16:0/OH)+matrix+H](+), and [sphingomyelin (SM)(d18:1/16:0)+H](+). Reduced expression of LPC(16:0/OH) in cancer tissue was an independent predictor of biochemical recurrence after radical prostatectomy. CONCLUSIONS: HR-MALDI-IMS showed that the expression of LPC(16:0/OH) and SM(d18:1/16:0) was lower in prostate cancer than in benign prostate epithelium. These differences in expression of phospholipids may predict prostate cancer aggressiveness, and provide new insights into lipid metabolism in prostate cancer.

Moist-condition Training for Cerebrovascular Anastomosis: A Practical Step after Mastering Basic Manipulations.

As cerebrovascular anastomosis is performed in moist conditions that may impede precise manipulations, surgeons must undergo extensive preoperative training. We developed a simple moist-condition training method. It involves placing a free-floating inner platform hosting an artery from a chicken wing in an outer container filled with tap water to just below the specimen. Trainees performed anastomosis under magnification. Training sessions mimicked difficulties encountered during operations such as poor visibility of the lumen and problems handling the sutures. A retrospective comparison of 100 wet- and 100 dry-condition training sessions for end-to-side anastomoses with 8 stitches showed that under moist condition the time required for the entire procedure was significantly longer (17.8 ± 2.1 vs. 15.3 ± 2.1 min, p < 0.01) and the incidence of wrong stitching was greater (0.38 vs. 0%, p = 0.04). In 8 cases after introducing moist-condition training, the time required in superficial temporal artery to middle cerebral artery bypass surgery was significantly shorter than 8 cases before introducing the training (32.3 ± 5.6 min vs. 48.3 ± 15.9 min, p = 0.01). Incidence of wrong stitches was less in cases after introducing moist-condition training (2.7 vs. 7.4%, p = 0.10). Those indicate that moist-condition training is a useful and practical step and a bridge between training for basic manipulations under dry conditions and actual surgery.

The C-terminal fragment of prostate-specific antigen, a 2331 Da peptide, as a new urinary pathognomonic biomarker candidate for diagnosing prostate cancer.

BACKGROUND AND OBJECTIVES: Prostate cancer (PCa) is one of the most common cancers and leading cause of cancer-related deaths in men. Mass screening has been carried out since the 1990s using prostate-specific antigen (PSA) levels in the serum as a PCa biomarker. However, although PSA is an excellent organ-specific marker, it is not a cancer-specific marker. Therefore, the aim of this study was to discover new biomarkers for the diagnosis of PCa. MATERIALS AND METHODS: We focused on urine samples voided following prostate massage (digital rectal examination [DRE]) and conducted a peptidomic analysis of these samples using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS(n)). Urinary biomaterials were concentrated and desalted using CM-Sepharose prior to the following analyses being performed by MALDI-TOF/MS(n): 1) differential analyses of mass spectra; 2) determination of amino acid sequences; and 3) quantitative analyses using a stable isotope-labeled internal standard. RESULTS: Multivariate analysis of the MALDI-TOF/MS mass spectra of urinary extracts revealed a 2331 Da peptide in urine samples following DRE. This peptide was identified as a C-terminal PSA fragment composed of 19 amino acid residues. Moreover, quantitative analysis of the relationship between isotope-labeled synthetic and intact peptides using MALDI-TOF/MS revealed that this peptide may be a new pathognomonic biomarker candidate that can differentiate PCa patients from non-cancer subjects. CONCLUSION: The results of the present study indicate that the 2331 Da peptide fragment of PSA may become a new pathognomonic biomarker for the diagnosis of PCa. A further large-scale investigation is currently underway to assess the possibility of using this peptide in the early detection of PCa.

The expression profile of phosphatidylinositol in high spatial resolution imaging mass spectrometry as a potential biomarker for prostate cancer.

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500-1000 was performed on optimal cutting temperature (OCT) compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS) showed that these molecules included 14 phosphatidylinositols (PIs), 3 phosphatidylethanolamines (PEs) and 3 phosphatidic acids (PAs). Among the PIs, the expression of PI(18:0/18:1), PI(18:0/20:3) and PI(18:0/20:2) were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA). The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

Modified wound dissection preserving the greater occipital nerve in foramen magnum decompression: a technique to reduce postoperative pain.

Patients undergoing foramen magnum decompression for Chiari malformation may experience severe postoperative pain in the area innervated by the greater occipital nerve (GON). We developed a modified dissection to lessen this pain. A midline skin incision was extended 2 cm in a cephalad direction to the inion and the skin was minimally retracted. After exposing the occipital bone, the semispinalis capitis and the trapezius muscles were detached subperiosteally in a caudal-to-cephalad direction. Consequently, the muscles and skin containing the GON were retracted in a single layer. We retrospectively compared the intensity of postoperative pain recorded on the visual analogue scale (VAS) by patients who underwent decompression using our (group A, n = 5) and the conventional layer-by-layer dissection technique (group B, n = 5). The VAS scores were not different on the day of surgery, but subsequently they fell faster in group A. Group A patients received a mild analgesic for a short period. Group B patients required a stronger analgesic for prolonged periods. Postoperative GON numbness/tenderness was observed only in group B. With respect to most evaluation criteria, the difference between the two groups was significant. Our anatomically rational dissection that protects the GON results in less postoperative pain.

[Dural scoring: an assistance technique for primary dural closure at craniotomy].

OBJECTIVE: At craniotomy the dura shrinks due to the drying effect of illumination and air exposure, rendering primary closure of this tissue difficult. We have developed a new technique, "dural scoring", that facilitates primary dural closure. PATIENTS AND METHODS: We used this technique in adults who underwent craniotomy when we encountered difficulties with primary dural closure of openings less than 5 mm in width. We placed scores along the edge of the opening on the surface of the dura (periosteal dura), taking care not to perforate the deep layer (meningeal dura). The dura relieved of tension expanded enough to be closed with sutures. RESULTS: This technique was successful in achieving primary dural closure in 53 patients who primarily underwent small supratentorial craniotomies. There were no procedure-related complications, e. g. cerebrospinal fluid leakages. CONCLUSIONS: Dural scoring is simple, requires no special instrumentation, and facilitates primary dural closure.

Anchoring of methylmethacrylate filler to the calvarium--technical note.

Implanted methylmethacrylate may be unexpectedly displaced due to poor adherence to the bone. We developed a simple technique to fix the material plugging the burr holes for use primarily in cosmetically important areas. At the closure of craniotomy, 2-3 small drill holes are made at the rim of the craniotomy burr hole. To address cranial defects in the pterional region, small holes are placed on the bone surface around the key burr hole. The holes extend into the diploic layer and have no parallel relationship. After fixation of the bone flap, a methylmethacrylate filler mixture is manually plugged into the burr hole and pushed into the small holes, thereby forming horns for secure fixation. None among over 100 patients developed an objectionable bulge attributable to displacement of the filler. Our technique requires no special instruments or materials and decreases the risk of cosmetic problems.

Radiofrequency radiation at 40 kHz induces hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease.

In the present study, we examined effects of radiofrequency (RF) radiation at 40 kHz on hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease, which is a heritable disease of copper metabolism in the liver. The activities of ALT and AST in serum of LEC rats exposed to RF radiation for 2 weeks were approximately 3.8-fold and 2-fold higher than those in serum of sham-exposed rats, respectively. Although there were no significant differences in hepatic copper contents between LEC rats exposed to RF radiation for 2 weeks and sham-exposed rats, copper contents in the kidney and serum of exposed LEC rats were approximately 4.2-fold and 12.9-fold higher than those in sham-exposed rats, respectively. Relative O2⁻-scavenging activities in the S-100 fraction of the liver of LEC rats exposed to RF radiation for 2 weeks were 1.6-fold higher than those in sham-exposed rats. No significant differences were observed in activities of AST and ALT in serum and relative O2⁻-scavenging activity in the S-100 fraction of the liver of normal control WKAH rats that were sham-exposed and exposed to RF radiation. No significant differences were observed in copper contents in the liver, kidney and serum of WKAH rats that were sham-exposed and exposed to RF radiation for 2 weeks. The results show that RF radiation at 40 kHz induced hepatic injury in LEC rats.

Repositioning of Cranial Bone Flaps Cut with a Diamond-Coated Threadwire Saw: 5-Year Experience with Cosmetic Cranioplasty without Fixation Devices.

Artificial fixation systems for cranial bone flaps have problems related to their materials and designs. We developed an alternative technique for supratentorial craniotomy that employs a diamond-coated threadwire saw (diamond T-saw), originally developed for spinal surgery, and reduces the bone gap for fitted bone flap fixation. The study subjects were 77 adults undergoing elective supratentorial craniotomy. After placing a burr hole at each corner of the craniotomy, we performed osteotomy between adjacent burr holes to approximately one-third of the length of the osteotomy with a craniotome; this leaves a bony bridge at each corner. The diamond T-saw was introduced between adjacent burr holes through the epidural space and a bridge was cut with reciprocating strokes. On closure, the bridge firmly supports the flap and only sutures are needed for fixation. Successful bone flap fixation was obtained in all followed-up cases. There were no technique-related complications such as dural laceration, flap displacement, or resorption. Our method is ideal for bone cuts in cosmetic cranioplasty; it is easy, safe, and inexpensive and avoids the need for flap fixation with artificial devices.

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4.

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells.

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

Video-assisted transaortic left ventricular thrombectomy and coronary artery bypass grafting.

A 59-year-old man with cardiac dysfunction was admitted to our hospital because of thrombus formation in the left ventricle 10 days following acute myocardial infarction. Echocardiography revealed evidence of two mobile thrombi, each measuring about 2 cm in diameter. Urgent coronary artery bypass grafting and video-assisted transaortic thrombectomy were performed without making a left ventricular incision to preserve his cardiac function. Endoscopy was useful for visualizing the anatomical structures in the left ventricular cavity.

The free radical scavenger edaravone rescues rats from cerebral infarction by attenuating the release of high-mobility group box-1 in neuronal cells.

Edaravone, a potent free radical scavenger, is clinically used for the treatment of cerebral infarction in Japan. Here, we examined the effects of edaravone on the dynamics of high-mobility group box-1 (HMGB1), which is a key mediator of ischemic-induced brain damage, during a 48-h postischemia/reperfusion period in rats and in oxygen-glucose-deprived (OGD) PC12 cells. HMGB1 immunoreactivity was observed in both the cytoplasm and the periphery of cells in the cerebral infarction area 2 h after reperfusion. Intravenous administration of 3 and 6 mg/kg edaravone significantly inhibited nuclear translocation and HMGB1 release in the penumbra area and caused a 26.5 +/- 10.4 and 43.8 +/- 0.5% reduction, respectively, of the total infarct area at 24 h after reperfusion. Moreover, edaravone also decreased plasma HMGB1 levels. In vitro, edaravone dose-dependently (1-10 microM) suppressed OGD- and H(2)O(2)-induced HMGB1 release in PC12 cells. Furthermore, edaravone (3-30 microM) blocked HMGB1-triggered apoptosis in PC12 cells. Our findings suggest a novel neuroprotective mechanism for edaravone that abrogates the release of HMGB1.