Serum proteomic identification and validation of two novel atherosclerotic aortic aneurysm biomarkers, profilin 1 and complement factor D.

BACKGROUND: Effective diagnostic biomarkers for aortic aneurysm (AA) that are detectable in blood tests are required because early detection and rupture risk assessment of AA can provide insights into medical therapy and preventive treatments. However, known biomarkers for AA lack specificity and reliability for clinical diagnosis. METHODS: We performed proteome analysis of serum samples from patients with atherosclerotic thoracic AA (TAA) and healthy control (HC) subjects to identify diagnostic biomarkers for AA. Serum samples were separated into low-density lipoprotein, high-density lipoprotein, and protein fractions, and the major proteins were depleted. From the proteins identified in the three fractions, we narrowed down biomarker candidates to proteins uniformly altered in all fractions between patients with TAA and HC subjects and evaluated their capability to discriminate patients with TAA and those with abdominal AA (AAA) from HC subjects using receiver operating characteristic (ROC) analysis. For the clinical validation, serum concentrations of biomarker candidates were measured in patients with TAA and AAA registered in the biobank of the same institute, and their capability for the diagnosis was evaluated. RESULTS: Profilin 1 (PFN1) and complement factor D (CFD) showed the most contrasting profiles in all three fractions between patients with TAA and HC subjects and were selected as biomarker candidates. The PFN1 concentration decreased, whereas the CFD concentration increased in the sera of patients with TAA and AAA when compared with those of HC subjects. The ROC analysis showed that these proteins could discriminate patients with TAA and AAA from HC subjects. In the validation study, these candidates showed significant concentration differences between patients with TAA or AAA and controls. PFN1 and CFD showed sufficient area under the curve (AUC) in the ROC analysis, and their combination further increased the AUC. The serum concentrations of PFN1 and CFD also showed significant differences between patients with aortic dissection and controls in the validation study. CONCLUSION: PFN1 and CFD are potential diagnostic biomarkers for TAA and AAA and measurable in blood samples; their diagnostic performance can be augmented by their combination. These biomarkers may facilitate the development of diagnostic systems to identify patients with AA.

Multiomics approach to identify novel biomarkers for dilated cardiomyopathy: Proteome and transcriptome analyses of 4C30 dilated cardiomyopathy mouse model.

Dilated cardiomyopathy (DCM) is an intractable disease, without any radical treatment option other than cardiac transplantation. Additionally, biomarkers to determine progressive staging are not yet available. Irrespective of the diversity of causative gene mutations, the phenotype of DCM is rather common. Therefore, it is plausible to determine DCM staging in terms of variations in protein and mRNA levels. In this study, we performed proteome and transcriptome analysis of the left ventricle of 4C30 DCM model mice showing mild and severe phenotypes at 12 and 24 weeks (wk) after birth, respectively. Proteomic analyses results showed 109 proteins that increased and 133 others that decreased among 1874 detected proteins. We selected biomarker candidates by confirming consistent alterations in protein levels at 12 and 24 wk, and mRNA levels at 12 wk, and narrowed these down based on the requirement that they should be detectable in blood. Finally, we selected six biomarker candidates based on sustained or augmented alteration at 24 wk and confirmed their definite alterations in the left ventricle by quantitative polymerase chain reaction, western blot analysis, and multiple reaction monitoring (MRM). To assess the validity of this strategy, we measured plasma concentrations of the six candidates by MRM method and identified two proteins (FTL1 and GRP78) that demonstrated significant elevation in the 4C30 mice plasma. Taken together, a multiomics strategy comparing tissue expression levels of proteins and mRNAs between diseased and control groups, with appropriate confirmation, is a promising approach for the discovery of new biomarkers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 491-502, 2016.

Identification of a chemical inhibitor for nuclear speckle formation: implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing.

Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A)(+) RNAs comprising mRNAs and poly (A)(+) non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A)(+) RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

[A case of an elderly woman with dementia in whom nutrition via gastrostomy was discontinued after considerable discussion according to the guidelines].

A 94-year-old woman with Alzheimer's disease had been receiving nourishment via gastrostomy for three years and in a state in which communication was not possible. Upon the family's request for consultation and advice, and after a considerable discussion according to the guidelines, the decision was made to stop the gastrostomy feeding. The patient subsequently coincidentally contracted influenza one week later and died due to pneumonia. Based on our experience of making the decision to stop nutritional supplementation via gastrostomy according to the guidelines, there are various issues, such as the psychological distress of the family, which cannot be resolved based on the guidelines.

[Usefulness of a manual on physician behavior at the time of death confirmation].

The behavior of a physician when confirming the death of a patient is thought to greatly affect the bereaved family. The required aspects of a physician's behavior after a patient's death are rarely included in physician education. Therefore, the few physicians who confirm the death of a patient should be conscious of the grief of the family members. A questionnaire survey was administered to nurses of a palliative care unit, and the findings showed that the behavior of an attending physician was different from that of other physicians when confirming death. We have prepared a manual that specifies the expected behavior of physicians confirming the death of patients to ensure that physicians other than the attending physician are also conscious of subsequent grief care for the bereaved family.

Comparative analysis of intercellular lipid organization and composition between psoriatic and healthy stratum corneum.

The lipid composition and organization of the stratum corneum (SC) in patients with psoriasis and healthy subjects were compared using X-ray diffraction, Fourier-transform infrared spectroscopy (FT-IR), and ultraperformance liquid chromatography, combined with time-of-flight mass spectrometry (UPLC-TOFMS). In healthy SC (HSC), SC lipids formed two lamellar phases (long and short periodicity phases). Hexagonal and orthorhombic hydrocarbon-chain packing were observed in the lateral lipid organization at 30 °C via X-ray diffraction. In HSC, the lamellar phases and the hydrocarbon-chain packing organizations changed with elevated temperatures and finally disappeared. In these behaviors, the high-temperature hexagonal hydrocarbon-chain packing organization, which appeared above the orthorhombic hydrocarbon-chain packing organization, transformed to the liquid phase at about 90 °C in HSC. In psoriatic SC (PSC), hexagonal hydrocarbon-chain packing organization disappeared at about 65 °C with elevated temperatures. No high-temperature hexagonal hydrocarbon-chain packing organization were observed in PSC during heating process. Disorder of the hydrocarbon-chain packing of SC lipids was observed in PSC via FT-IR. In UPLC-TOFMS, free fatty acid (FFA) and ceramide (CER) compositions differed between patients with PSC and HSC. Specifically, the levels of ultra-long chain fatty acids containing CER and phytosphingosine-containing CER were decreased, while those of sphingosine and dihydrosphingosine-containing CER and unsaturated FFA were increased in PSC. Furthermore, FFA and CER carbon chain lengths decreased in patients with PSC. These results suggest that the alteration of SC lipid composition and the reduction of carbon chain lengths in PSC lowered the structural transformation temperature, thereby reducing barrier function.

A small molecule inhibitor of p53-inducible protein phosphatase PPM1D.

PPM1D is a p53-inducible Ser/Thr protein phosphatase. PPM1D gene amplification and overexpression have been reported in a variety of human tumors, including breast cancer and neuroblastoma. Because the phosphatase activity of PPM1D is essential for its oncogenic role, PPM1D inhibitors should be viable anti-cancer agents. In our current study, we showed that SPI-001 was a potent and specific PPM1D inhibitor. SPI-001 inhibited PPM1D phosphatase activity in PPM1D-overexpressing human breast cancer cells and increased phosphorylation of p53. Furthermore, SPI-001 suppressed cell proliferation by inducing apoptosis. Our present study suggested that SPI-001 was a potential lead compound in developing anti-cancer drugs.

Correlation in the densities of augmented and existing bone in guided bone augmentation.

OBJECTIVES: Our previous work used a rabbit experimental model to investigate the effectiveness of guided bone augmentation (GBA). Although a density similar to that of existing bone is required for successful bone augmentation, few studies have compared the densities of augmented and existing bone. The purpose of the present study was to investigate the correlation in the densities of existing and augmented bone following GBA in rabbit calvaria. MATERIAL AND METHODS: The calvaria of 18 adult male Japanese white rabbits were exposed. A circular groove and nine small holes were drilled into the cortical surface of each left parietal bone. A customized, standardized, hemispherical titanium cap was press-fitted into each groove. Six animals were sacrificed after each healing period of 1, 3, and 6 months and histomorphometric analyses were conducted. RESULTS: Significant increases were observed in the area of augmented bone between 1 and 6 months (62.7 ± 21.6% vs. 93.4 ± 3.9%). In contrast, no significant differences among healing periods were observed in the density of augmented or existing bone. Regression analysis demonstrated a significant positive correlation between the densities of augmented and existing bone; the strength of this correlation increased with the length of healing (R(2) =0.97). CONCLUSIONS: These results suggest that the area of augmented bone increases significantly with the length of healing, filling the occlusive space after 6 months, and that the density of augmented bone depends on that of the existing bone, such that augmented bone has a density about half that of the existing bone.

Relation between connective tissue growth factor and cardiac sympathetic nerve activity in heart failure in DCM patients.

Cardiac fibrosis is an important process of myocardial remodeling. Connective tissue growth factor (CTGF) is a cytokine that plays a key role in the occurrence of progressive fibrosis and excessive scarring. CTGF levels are increased in the failing heart. In addition, sympathetic nerve activity is enhanced in the failing heart, and exacerbates heart failure. To clarify the relation between cardiac sympathetic nerve activity and CTGF, we studied 35 (M/F = 28/7 patients) aged 57 ± 15 years with dilated cardiomyopathy (DCM). Cardiac sympathetic nerve activity was estimated from the total defect score (TDS) and from the H/M ratio and washout rate (WR) on I-123-MIBG imaging. Cardiac symptoms (NYHA class), exercise capacity (specific activity scale: SAS), brain natriuretic peptide (BNP), hemodynamics, and CTGF were assessed. There was a significant correlation between the CTGF and WR on I-123-MIBG (r = 0.45, P = 0.008). Also, a higher plasma CTGF level was associated with a lower SAS score (r = 0.51, P = 0.002), but not the TDS, H/M ratio, or BNP concentration. Moreover, a higher NYHA class and pulmonary artery wedge pressure were associated with a higher plasma CTGF level. The plasma CTGF level can be strongly related with cardiac sympathetic nerve activity in heart failure in DCM patients.

Darier disease successfully treated with a topical agent containing vitamin A (retinyl palmitate), vitamin E, and urea.

Darier disease (DD), also called keratosis follicularis, is an autosomal dominant hereditary keratinization disorder that manifests as keratotic papules with plaques in seborrheic areas. There are no validated curative treatments for DD, with the majority of cases treated symptomatically. We report the efficacy of a topical over-the-counter agent which contains retinyl palmitate, vitamin E, and urea for a patient with DD. A 13-year-old girl had brown papules on her scalp, neck, shoulders, and axillae since entering elementary school. A skin biopsy revealed hyperkeratosis, suprabasal acantholysis, and dyskeratosis manifested as corps ronds and grains in the epidermis. Sanger sequencing found the previously reported heterozygous mutation c.1484C>T in ATP2A2. The application of an over-the-counter topical agent containing retinyl palmitate 2750 µg/g (5000 IU/g), tocopheryl acetate 20 mg/g, urea 200 mg/g, and monoammonium glycyrrhizinate 5 mg/g twice daily for 2 months improved the papules without serious adverse events. Oral or topical aromatic vitamin A analogs (retinoids) are often used to treat DD. However, several adverse events are associated with retinoid treatment, and many patients only undergo their intermittent use or discontinue the treatments. Retinyl palmitate is more stable and has a lower irritative profile than other retinoic acids. When applied topically, however, retinyl palmitate cannot penetrate the skin as well as retinol can. Some reports have noted that vitamin E increases the biological availability of vitamin A and that urea helps mechanical percutaneous drug delivery. Our case suggests that retinyl palmitate has a sufficient therapeutic effect when combined with vitamin E and urea. In conclusion, we propose that topical agents containing retinyl palmitate, vitamin E, and urea might have a satisfactory effect on the skin lesions of DD patients, without the serious risks of adverse events.

A new susceptibility index to predict the risk of severe herpes zoster-associated pain: A Japanese regional population-based cohort study, the Shizuoka study.

BACKGROUND: Approximately 7-20% of patients with herpes zoster (HZ) develop zoster-associated pain (ZAP). ZAP not only impairs quality of life and psychological well-being, but also can reduce work effectiveness, which has negative economic effects. Reports of ZAP risk factors are inconsistent. OBJECTIVE: To confirm risk factors for the development of severe ZAP in HZ patients in Japan using a large-scale database, the Shizuoka Kokuho Database. METHODS: A population-based cohort study using the Shizuoka Kokuho Database was conducted. Of 792,647 patients, 7491 (0.95%) experienced "severe ZAP" (as defined in this study). We developed a ZAP risk prediction scoring system by identifying risk factors using logistic regression analysis of several candidate risk factors for severe ZAP: age, sex, seasonality, and presence of comorbidities (using the Charlson comorbidity index), excluding HIV/AIDS. RESULTS: We identified peripheral vascular disease and the onset from October to December as novel risk factors for severe ZAP, in addition to the previously reported risk factors of age and comorbidities (cerebral vascular disease, chronic pulmonary disease, rheumatic disease, peptic ulcer, liver disease, diabetes, and malignant neoplasms with/without metastasis). In contrast, dementia was found to reduce ZAP risk. We developed a susceptibility index to predict the risk of ZAP. CONCLUSION: We newly demonstrated that peripheral vascular disease and the onset from October to December are ZAP risk factors. Our comorbidity findings support previous observations. The susceptibility index proposed here provides a new approach to the prevention of ZAP using early intervention for high-risk patients.

Late-onset development of psoriasis in Japan: a population-based cohort study.

BACKGROUND: Evidence of factors associated with psoriasis from large population-based cohort studies is scarce. OBJECTIVE: We aimed to explore the risk factors of late-onset psoriasis. METHODS: This study included 487,835 Japanese participants aged 40-107 years, who were followed prospectively from 2012 to 2018 using individually linked databases between annual health checkups and medical claims. RESULTS: During the study period, 2793 patients (0.57%) newly developed psoriasis; 13.8% had moderate-to-severe psoriasis. In the multivariate analysis, factors associated with psoriasis onset were age (hazard ratio [HR] 1.11 {95% confidence interval [CI]: 1.06-1.16}), male sex (HR: 1.11 [95% CI: 1.02-1.21]), body mass index (HR: 1.09 [95% CI: 1.05-1.14]), smoking (HR: 1.46 [95% CI: 1.31-1.63]), not exercising ≥1 hour per week (HR: 1.13 [95% CI: 1.05-1.22]), and gamma-glutamyl transpeptidase (HR: 1.04 [95% CI: 1.01-1.06]). When we used weight increment of ≥10 kg since the age of 20 years instead of body mass index in the multivariate model, this was also a risk factor (HR: 1.12 [95% CI: 1.04-1.21]). LIMITATIONS: This study targeted people aged >40 years, thereby narrowing the search to the risk factors of late-onset psoriasis. CONCLUSION: We showed that increasing age, male sex, body mass index, smoking, low physical activity, weight gain, and gamma-glutamyl transpeptidase are associated with late-onset development of psoriasis and revealed a relationship between liver dysfunction and psoriasis development.

Promiscuous interaction between gold-specific T cells and APCs in gold allergy.

Gold compounds clinically used as immunomodulators have high potential to evoke hypersensitivity reactions as an adverse effect. To explore the mechanism of gold allergy, we immunologically characterized T cells infiltrating skin rashes and generated 44 gold-specific T cell clones and lines from a rheumatoid arthritis patient who developed skin rashes and systemic symptoms after gold treatment. CD4(+) and CD8(+) cells predominantly infiltrating the skin rashes and some of the T cell clones and lines shared common Vbetas. These cells exhibited Th0-like, Th2-like, and Tc1-like cytokine profiles, and showed chemotactic activities for thymus and activation-regulated chemokine and IFN-gamma-inducible protein-10 corresponding to the cytokine profiles. T cell recognition of gold consisted of MHC-restricted and MHC-independent pathways. Blocking studies with anti-MHC Abs indicated that the groove of MHC in APCs, where Ags should ordinarily be settled, did not serve as a conjugating site of gold for these T cells in certain cases. These observations raise the possibility that gold-specific CD4(+) and CD8(+) T cells and APCs promiscuously interact under stimulation with gold, resulting in various clinical manifestations in gold allergy.

[Parotid gland cancer treatment with facial nerve preservation].

Based on clinical features in 34 cases of parotid gland cancer treated between January 1997 and December 2007, tumor, node, and metastasis classification found 5 subjects to be T1, 12 to be T2, 7 to be T3, and 10 to be T4a. Of these, 25 were staged for N0, 3 for N1, and 6 for N2. Histopathologically, 10 different tumor types were observed, with carci-noma ex pleomorphic adenoma the most common. Preoperative fine-needle aspiration cytology (FNAC) of parotid gland tumor was done on 126 and cytological findings compared to histopathologic diagnoses of surgically resected specimens. Sensitivity for malignancy was 76.0%, specificity 95.4%, and overall accuracy 91.1%. Six malignant tumors were diagnosed as benign and 4 benign as malignant by FNAC, indicating that FNAC results alone may not be sufficient for diagnosing malignancy definitively. Among subjects, 25 underwent surgical resection, with the facial nerve preserved in 15 of 29. Postoperative radiotherapy was indicated if lymph node metastasis, intermediate or high-grade malignancy, or positive margins were seen. Of 15 subjects undergoing postoperative radiotherapy, 3 experienced recurrence. For unresectable tumors, concurrent chemoradiotherapy was used for 3 subjects and 2 underwent radiotherapy alone, with all 5 current survivors. The 5-year overall survival (OS) was 87.4% and progression-free survival (PFS) 71.4%. In terms of 5-year PFS, significant differences were seen between stage I/II (91.7%) and stage III/IW (51.6%) (P=0.032), and between N0 (86.2%) and N+ (38.1%) (P=0.002).

Association of dry skin with intercellular lipid composition of stratum corneum after erlotinib administration.

PURPOSE: Erlotinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, causes skin disorders such as dry skin, which impairs the skin barrier function. Stratum corneum (SC) lipids play an important role in skin barrier function; therefore, this study aimed to investigate the relationship between erlotinib-related dry skin and changes in the intercellular lipid composition and structure of the SC. METHODS: Overall, 21 patients with non-small lung cancer were enrolled in this study. All patients received 150 mg/day erlotinib orally. A SC sample of each patient was collected from the inner forearm using the tape stripping method on days 0, 7, 14, 28, and 56 after erlotinib administration. The intercellular lipid components of ceramide (CER), free fatty acid (FFA), and cholesterol sulfate (CS) in samples extracted from the tape were analyzed using liquid chromatography/time-of-flight/mass spectrometry. SC samples from six healthy subjects were collected as controls on days 0, 28 and 56 and analyzed similarly. RESULTS: Although total CER and FFA levels were not changed after erlotinib administration, the levels of CER subclasses [AP] and [AH] and hydroxy FFA, which are structural components of CER subclass [A], decreased. In contrast, the CS levels increased after erlotinib administration. Moreover, higher CS levels in the SC correlated with the clinical condition of dry skin. No changes were observed in the SC lipid composition in healthy subjects. CONCLUSION: Erlotinib-related dry skin was associated with a higher CS level in the SC.

Discovery of novel biomarkers for atherosclerotic aortic aneurysm through proteomics-based assessment of disease progression.

Since aortic aneurysms (AAs) are mostly asymptomatic, but they have a high mortality rate upon rupture, their detection and progression evaluation are clinically important issues. To discover diagnostic biomarkers for AA, we performed proteome analysis of aortic media from patients with thoracic atherosclerotic AA (TAAA), comparing protein levels between the aneurysm and normal tissue areas. After hierarchical clustering analysis of the proteome analysis data, tissue samples were classified into three groups, regardless of morphological features. This classification was shown to reflect disease progression stage identified by pathological examination. This proteomics-based staging system enabled us to identify more significantly altered proteins than the morphological classification system. In subsequent data analysis, Niemann-Pick disease type C2 protein (NPC2) and insulin-like growth factor-binding protein 7 (IGFBP7) were selected as novel biomarker candidates for AA and were compared with the previously reported biomarker, thrombospondin 1 (THBS1). Blood concentrations of NPC2 and IGFBP7 were significantly increased, while THBS1 levels were decreased in TAAA and abdominal atherosclerotic AA patients. Receiver operating characteristic analysis of AA patients and healthy controls showed that NPC2 and IGFBP7 have higher specificity and sensitivity than THBS1. Thus, NPC2 and IGFBP7 are promising biomarkers for the detection and progression evaluation of AA.

Publisher Correction: Discovery of novel biomarkers for atherosclerotic aortic aneurysm through proteomics-based assessment of disease progression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.