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Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation. With these unique features, targeted proteomics can seamlessly transfer cancer biomarker candidate proteins from the discovery phase to the verification and validation phases, thereby resulting in an accelerated cancer biomarker pipeline. Furthermore, understanding the basic principles, advantages, and disadvantages is necessary to effectively utilize targeted proteomics in cancer biomarker pipelines. This review aimed to introduce the technical principles of targeted proteomics for cancer biomarker verification and validation. [ABSTRACT FROM AUTHOR]

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents. [ABSTRACT FROM AUTHOR]

Genes encoding human β-type globin undergo a developmental switch from embryonic to fetal to adult-type expression. Mutations in the adult form cause inherited hemoglobinopathies or globin disorders, including sickle cell disease and thalassemia. Some experimental results have suggested that these diseases could be treated by induction of fetal-type hemoglobin (HbF). However, the mechanisms that repress HbF in adults remain unclear. We found that the LRF/ZBTB7A transcription factor occupies fetal γ-globin genes and maintains the nucleosome density necessary for γ-globin gene silencing in adults, and that LRF confers its repressive activity through a NuRD repressor complex independent of the fetal globin repressor BCL11A. Our study may provide additional opportunities for therapeutic targeting in the treatment of hemoglobinopathies. [ABSTRACT FROM AUTHOR]

Background: Aldehyde dehydrogenase (ALDH) is highly expressed in stem/progenitor cells in various tissues, and cell populations with high ALDH activity (ALDHbr) are associated with tissue repair. However, little is known about lung-resident ALDHbr. This study was performed to clarify the characteristics of lung-resident ALDHbr cells and to evaluate their possible use as a tool for cell therapy using a mouse model of bleomycin-induced pulmonary fibrosis. Methods: The characteristics of lung-resident/nonhematopoietic (CD45−) ALDHbr cells were assessed in control C57BL/6 mice. The kinetics and the potential usage of CD45−/ALDHbr for cell therapy were investigated in bleomycin-induced pulmonary fibrosis. Localization of transferred CD45−/ALDHbr cells was determined using mCherry-expressing mice as donors. The effects of aging on ALDH expression were also assessed using aged mice. Results: Lung CD45−/ALDHbr showed higher proliferative and colony-forming potential than cell populations with low ALDH activity. The CD45−/ALDHbr cell population, and especially its CD45−/ALDHbr/PDGFRα+ subpopulation, was significantly reduced in the lung during bleomycin-induced pulmonary fibrosis. Furthermore, mRNA expression of ALDH isoforms was significantly reduced in the fibrotic lung. When transferred in vivo into bleomycin-pretreated mice, CD45−/ALDHbr cells reached the site of injury, ameliorated pulmonary fibrosis, recovered the reduced expression of ALDH mRNA, and prolonged survival, which was associated with the upregulation of the retinol-metabolizing pathway and the suppression of profibrotic cytokines. The reduction in CD45−/ALDHbr/PDGFRα+ population was more remarkable in aged mice than in young mice. Conclusions: Our results strongly suggest that the lung expression of ALDH and lung-resident CD45−/ALDHbr cells are involved in pulmonary fibrosis. The current study signified the possibility that CD45−/ALDHbr cells could fnd application as novel and useful cell therapy tools in pulmonary fibrosis treatment. [ABSTRACT FROM AUTHOR]

2',4'-Bridged modifications such as 2'-O,4'-C-methylene-bridged nucleotides (LNAs) and 2'-O,4'-C-ethylene-bridged nucleotides (ENAs) provide high binding affinity for duplex formation. Stabilization by the introduction of the bridged nucleic acids is considered to be due to pre-organization. In this study, we found that the introduction of 2',4'-C-bridged 2'-deoxynucleotides (CRNs; Conformationally Restricted Nucleotides) into DNA/RNA duplexes leads to destabilization, as opposed to the previously accepted notion that 2',4'-bridged modifications always lead to stabilization. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T. [ABSTRACT FROM AUTHOR]

An intermediate filament protein, Nestin, is known as a neural stem/progenitor cell marker. It was shown to be required for the survival and self-renewal of neural stemcells according to the phenotypes of Nestin knockoutmice. Nestin expression has also been reported in vascular endothelial cells, and we recently reported Nestin expression in proliferating endothelial progenitor cells, but not in mature endothelial cells. Using quantitative phosphoproteome analysis, we studied differences in phosphorylation levels between CNS Nestin in adult neural stem cells and vascular Nestin in adult bone-marrow-derived endothelial progenitor cells.We detected 495 phosphopeptides in the cell lysates of adult CNS stem/progenitor cells and identified 11 significant phosphorylated amino acid residues in the Nestin protein. In contrast, endothelial progenitor cells showed no significant phosphorylation of Nestin. We also measured neoplastic endothelial cells of the mouse brain and identified 13 phosphorylated amino acid residues in the Nestin protein. Among the 11 phosphorylated amino acids of adult CNS Nestin, five (S565, S570, S819, S883, and S886) were CNS Nestin-specific phosphorylation sites. Detection of the CNS-specific phosphorylation sites in Nestin, for example, by a phospho-specific Nestin antibody, may allow the expression of CNS Nestin to be distinguished from vascular Nestin. [ABSTRACT FROM AUTHOR]

STUDY DESIGN.: A retrospective, age- and sex-matched radiographic study. OBJECTIVES.: To investigate the spinopelvic alignment in patients with osteoarthosis of the hip (HOA) and those with low back pain (LBP) and to determine the characteristics and differences in both groups. SUMMARY OF BACKGROUND DATA.: Hip-spine syndrome, first described by Offierski and MacNab, is quite an important pathology when treating patients with pain in their low back and lower extremities. However, despite it being a well-known entity, few papers have adequately investigated and assessed the spinopelvic alignment in patients with hip-spine syndrome. METHODS.: Sagittal and coronal spinopelvic alignments were investigated in 150 patients with HOA and 150 with LBP using radiographs of the whole spine in both anteroposterior and lateral views. Parameters measured in this study were lumbar lordosis (LL), sacral slope (SS), the shift of the sagittal C7 plumb line, pelvic incidence (PI), and pelvic tilt (PT) on the lateral radiographs. On the anteroposterior (AP) films, lumbar scoliosis, pelvic obliquity, leg length discrepancy, the shift of the coronal C7 plumb line, and Sharp angle were measured. These parameters were compared between the two groups. In patients with HOA, the relationships between Sharp angle and other parameters were also analyzed to clarify the possible influence of sagittal and coronal spinopelvic alignments on HOA without acetabular dysplasia. RESULTS.: LL, SS, PI, and PO were found to be less in patients with LBP compared with those with HOA, and there was no significant difference in LS between the two groups. PI was significantly greater in HOA patients and strongly correlated to PT, SS, and LL (i.e., as the PI increased so did the PT, SS, and LL). Sharp angles were also significantly greater in HOA patients and strongly correlated to age, LL and SS (i.e., as Sharp angles increased so did LL and SS); however, age decreased in the hip patients. CONCLUSIONS.: These findings suggest that higher PI in the younger individual may contribute to the development of HOA in later life without both lumbar kyphosis and acetabular dysplasia because of the anterior uncovering of the acetabulum. More investigation will be expected to analyze the spinopelvic alignment in patients with hip spine syndrome. [ABSTRACT FROM AUTHOR]

Study Design: Retrospective longitudinal cohort study.Objective: To investigate the association between diffuse idiopathic skeletal hyperostosis (DISH) and reoperation in patients treated surgically for lumbar spinal stenosis (LSS) in long-term results.Summary Of Background Data: Few studies have evaluated DISH as a potential risk factor of poor surgical results for LSS.Methods: This study included 1063 responders to a postoperative postal survey out of 2363 consecutive patients who underwent surgery for LSS between 2002 and 2010. The survey included questions about reoperations performed at another hospital and the patient-reported outcomes. DISH was evaluated by preoperative standing whole-spine radiographs. We investigated DISH as a predictor of reoperation and characteristics of poor outcomes in patients with DISH. We also assessed selection bias by examining the differences between responders and nonresponders to a postal survey.Results: Reoperations were performed in a total of 115 patients (10.8%) within an average of 8.6 years after the initial surgeries. Patients who only had DISH were not associated with reoperation; however, reoperations were performed significantly more often in patients with DISH extended to the lumbar segment (L-DISH) than in patients without (22% and 7.3%, respectively; P < 0.001). Cox analysis showed that L-DISH was one of the significant independent predictors for reoperation (hazard ratio 2.05, P = 0.009). Surgery-free survival was significantly shorter in patients with L-DISH than in those without (P = 0.005). The cause of reoperation did not differ between the patients with and without L-DISH. Several factors, but not L-DISH, were significantly associated with responders to the survey.Conclusion: L-DISH was independently associated with reoperation for LSS. The decreased number of lumbar mobile segments due to L-DISH might lead to unfavorable outcomes. Careful follow-up of patients is needed after surgery for LSS with L-DISH.Level Of Evidence: 3. [ABSTRACT FROM AUTHOR]

We developed a simplified and sensitive method to identify Alzheimer's disease (AD) biomarker candidates by a quantitative and targeted proteomic analysis (combination of liquid chromatography tandem mass spectrometry and multiplexed-multiple reaction monitoring/ selected reaction monitoring analysis) of culture media from neurons differentiated from induced pluripotent stem cells (iPSCs) established from AD patients. We found that alpha-1-acid glycoprotein (ORM1) was decreased in the culture media of AD-iPSC-derived neurons, consistent with previous observations for AD patient cerebrospinal fluid, thus validating our new strategy. Moreover, our method is applicable for identifying biomarker candidates for other neurodegenerative disorders using patient-derived iPSCs. [ABSTRACT FROM AUTHOR]

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor that secretes various angiogenic factors. The main inhibitor of plasminogen activators, PAI-1 (SERPINE1), has been implicated in tumor progression and angiogenesis, and high PAI-1 expression has been associated with poor prognosis in MPM patients. In this study, we examined the antiangiogenic effects of PAI-1 inhibition in MPM. We administered the PAI-1 inhibitor, SK-216, to orthotopic mouse models in which MPM cells expressing high levels of VEGF (VEGFA) or bFGF (FGF2) were intrapleurally transplanted. SK-216 administration reduced tumor weights and the degree of angiogenesis in intrapleural tumors, irrespective of their angiogenic expression profiles. In addition, a combination of SK-216 and the chemotherapeutic agent cisplatin significantly reduced tumor weights compared with monotherapy, prolonging the survival of animals compared with cisplatin treatment alone. Furthermore, SK-216 inhibited migration and tube formation of cultured human umbilical vein endothelial cells induced by various angiogenic factors known to be secreted by MPM. These findings suggest that PAI-1 inactivation by SK-216 may represent a general strategy for inhibiting angiogenesis, including for the treatment of MPM. [ABSTRACT FROM AUTHOR]

Background. Recent reports indicate thatmatrixmetalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential disease markers of idiopathic pulmonary fibrosis (IPF). The objective of this study was to perform direct comparisons of these two biomarkers with three well-investigated serum markers of IPF, Krebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D. Methods. The serum levels of MMP-7, CCL18, KL-6, SP-A, and SP-D were evaluated in 65 patients with IPF, 31 patients with bacterial pneumonia, and 101 healthy controls. The prognostic performance of these five biomarkers was evaluated in patients with IPF. Results. The serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared to those in patients with bacterial pneumonia and in the healthy controls. Multivariate survival analysis showed that serumMMP-7 and KL-6 levels were independent predictors in IPF patients. Moreover, elevated levels of both KL-6 andMMP-7 were associated with poorer survival rates in IPF patients, and the combination of both markers provided the best risk discrimination using the C statistic. Conclusions. The present results indicated that MMP-7 and KL-6 were promising prognostic markers of IPF, and the combination of the two markers might improve survival prediction in patients with IPF. [ABSTRACT FROM AUTHOR]

Study Design. Matched case-control study. Objective. To identify factors other than a multilevel procedure that increase the risk of symptomatic postoperative spinal epidural hematoma (SEH). Summary of Background Data. Postoperative SEH is a potentially devastating complication of spinal surgery. Previous studies that reported risk factors for postoperative SEH all identified a multilevel procedure as a risk factor, but the other risk factors remain unclear. Methods. Patients who developed postoperative SEH requiring surgical evacuation were identified from database. Each patient was matched with 3 controls who underwent spinal decompression at the same number of levels in the same part of the spine by the same surgeon during the preceding or following year. Multiple logistic regression analysis was performed to identify the risk factors for postoperative SEH to obtain adjusted odds ratios with 95% confidence intervals. Clinical outcomes after evacuation were investigated separately divided with or without severe paralysis or time until the second surgery. Results. Postoperative SEH evacuation was performed after 32 of 8250 (0.39%) spinal decompression procedures. The incidence was significantly higher after thoracic procedures (2.41%) than after cervical (0.21%) or lumbar (0.39%) procedures. Multivariate analysis identified a 50 mm Hg or greater increase in systolic blood pressure after extubation (adjusted odds ratio: 3.22, 95% confidence interval: 1.22-8.51) and higher body mass index (adjusted odds ratio 1.15, 95% confidence interval: 1.01-1.31) as risk factors. Among 14 patients with severe paralysis due to postoperative SEH, those who underwent evacuation within 24 hours of the onset had a significantly better improvement in clinical outcome and Frankel grade than did those after 24 hours. Conclusion. A 50 mm Hg or greater increase in systolic blood pressure after extubation and high body mass index were identified as risk factors for SEH. Appropriate blood pressure control especially at the end of surgery is important for the prevention of postoperative SEH, particularly in obese patients. [ABSTRACT FROM AUTHOR]

Hip range of motion (ROM) may be an important preoperative variable, however, measurement of hip ROM can be affected by various factors. The purposes of this study were to compare conventional preoperative ROM measurements with those measurements obtained under general anaesthesia, and to better define the associations between preoperative hip ROM, and demographic, functional, and diagnostic variables. Conventional preoperative hip ROM and ROM under general anaesthesia were prospectively measured in 471 hips. Harris pain score, the Crowe classification, and the diagnosis were also investigated. The hip ROM in all directions under general anaesthesia was significantly greater than conventional preoperative hip ROM. There were correlations between conventional preoperative hip ROM and the Harris hip pain score. Hip ROM with Crowe type I deformity under anaesthesia was significantly greater than in hips with type II, III or IV. ROM under anaesthesia of osteonecrosis hips was significantly greater than osteoarthritis and dislocated hips. Conventional preoperative ROM in men was greater in flexion and external rotation compared to women. Internal rotation motion in women in both conventional preoperative ROM and ROM under anaesthesia was greater than in men. Hip pain, Crowe classification and diagnosis could influence preoperative hip ROM. Measurement of hip ROM under general anaesthesia could better reflect true ROM compared to measurement without anaesthesia. [ABSTRACT FROM AUTHOR]