Proteomic profile of differentially expressed plasma proteins from dystrophic mice and following suberoylanilide hydroxamic acid treatment

Purpose: Histone Deacetylase Inhibitors (DI) ameliorates dystrophic muscle regeneration restoring muscular strength in the mdx mouse model of Duchenne muscular dystrophy (DMD). The further development of these compounds as drugs for DMD treatment is currently hampered by the lack of knowledge about DIs effect in large dystrophic animal models and that of suitable biomarkers to monitor their efficacy. Experimental design: In this study we applied proteomic analysis to identify differentially expressed proteins present in plasma samples from mdx mice treated with the Suberoylanilide hydroxamic acid (SAHA) and relative normal controls (WT). Results: Several differentially expressed proteins were identified between untreated wild type and mdx mice. Among these, fibrinogen, epidermal growth factor 2 receptor, major urinary protein and glutathione peroxidase 3 (GPX3) were constitutively up‐regulated in mdx, while complement C3, complement C6, gelsolin, leukaemia inhibitory factor receptor (LIFr), and alpha 2 macroglobulin were down‐regulated compared to WT mice. SAHA determined the normalization of LIFr and GPX3 protein level while apoliprotein E was de novo up‐regulated in comparison to vehicle‐treated mdx mice. Conclusions and clinical relevance: Collectively, these data unravel potential serological disease biomarkers of mdx that could be useful to monitor muscular dystrophy response to DI treatment.     

Designing of multi-targeted molecules using combination of molecular screening and in silico drug cardiotoxicity prediction approaches

We have previously investigated and reported a set of phenol- and indole-based derivatives at the binding pockets of carbonic anhydrase isoenzymes using in silico and in vitro analyses. In this study, we extended our analysis to explore multi-targeted molecules from this set of compounds. Thus, 26 ligands are screened at the binding sites of 229 proteins from 5 main enzyme family classes using molecular docking algorithms. Derived docking scores are compared with reported results of ligands at carbonic anhydrase I and II isoenzymes. Results showed potency of multi-targeted drugs of a few compounds from investigated ligand set. These promising ligands are then tested in silico for their cardiotoxicity risks. Results of this work can be used to improve the desired effects of these compounds by molecular engineering studies. In addition these results may lead to further investigation of studied molecules by medicinal chemists to explore different therapeutic aims.     

Two‐dimensional gel electrophoresis of apolipoprotein C‐III and other serum glycoproteins for the combined screening of human congenital disorders of O‐ and N‐glycosylation

Congenital disorders of glycosylation (CDG) are inherited diseases that can affect not only the N‐glycan (e.g. CDG type I and II) but also the O‐glycan biosynthesis pathway. In the absence of specific clinical symptoms, there is a need for a reliable biological screening of these two groups of CDG. Using a few microlitres of human serum, 2‐DE and immunoblotting were applied to the separation and simultaneous detection of the isoforms of the O‐glycosylated protein apolipoprotein C‐III (apoC‐III) and of four N‐glycosylated proteins, namely alpha‐antitrypsin, alpha‐1 acid glycoprotein, haptoglobin and transferrin. For the study of O‐glycosylation, this technique allowed the reliable separation of the three fractions of apoC‐III and the determination of normal percentage values in an adult population. Concerning N‐glycosylation, the study of serum samples from patients with CDG type Ia revealed marked abnormalities systematically affecting the four 2‐DE separated N‐linked glycoproteins. 2‐DE coupled to immunoblotting using a mixture of specific antibodies could be easily and reliably employed for the combined screening of both N‐ and O‐glycosylation disorders in humans.     

Efficient and bright blue‐emitting phosphorescent materials

Abstract— A new type of ancillary ligand for blue‐emitting heteroleptic iridium complexes has been successfully developed. New ligands, 3‐(trifluoromethyl)‐5‐(pyridin‐2‐yl)‐1,2,4‐triazolate and 5‐(pyridin‐2‐yl)‐tetrazolate, show stronger blue‐shifting power than that of the picolate of FIrpic [iridium (III) bis(4,6‐difluorophenylpyridinato)picolate]. Organic light‐emitting diodes (OLEDs) fabricated with a new complex, FIrtaz [iridium (III) bis(4,6‐difluorophenylpyridinato)(5‐(pyridine‐2‐yl)‐1,2,4‐triazolate) or FIrN4 [(iridium (III) bis(4,6‐difluorophenylpyridinato)(5‐(pyridin‐2‐yl)‐tetrazolate], as the blue dopant in the host of mCP [1,3‐ bis(9‐carbazolyl)benzene], exhibit near‐saturated blue electrophosphorescence with Commision Internale de l'Eclairage (CIE^x,y) coordinates of (0.14, 0.18) and (0.15, 0.24), respectively.     

Application of clustering analyses to the diagnosis of Huntington disease in mice and other diseases with well-defined group boundaries

Nuclear magnetic resonance (NMR) spectroscopy has emerged as a technology that can provide metabolite information within organ systems in vivo. In this study, we introduced a new method of employing a clustering algorithm to develop a diagnostic model that can differentially diagnose a single unknown subject in a disease with well-defined group boundaries. We used three tests to assess the suitability and the accuracy required for diagnostic purposes of the four clustering algorithms we investigated (K-means, Fuzzy, Hierarchical, and Medoid Partitioning). To accomplish this goal, we studied the striatal metabolomic profile of R6/2 Huntington disease (HD) transgenic mice and that of wild type (WT) mice using high field in vivo proton NMR spectroscopy (9.4 T). We tested all four clustering algorithms (1) with the original R6/2 HD mice and WT mice, (2) with unknown mice, whose status had been determined via genotyping, and (3) with the ability to separate the original R6/2 mice into the two age subgroups (8 and 12 weeks old). Only our diagnostic models that employed ROC-supervised Fuzzy, unsupervised Fuzzy, and ROC-supervised K-means Clustering passed all three stringent tests with 100% accuracy, indicating that they may be used for diagnostic purposes.     

In pursuit of B-cell synovial autoantigens in rheumatoid arthritis: Confirmation of citrullinated fibrinogen, detection of vimentin, and introducing carbonic anhydrase as a possible new synovial autoantigen

We aimed to investigate potential synovial autoantigens in rheumatoid arthritis (RA) that could trigger the induction of B‐cell autoantibodies. Total protein extract of synovial tissue obtained from seven RA patients was pooled and separated by 1‐DE and 2‐DE. The corresponding blots were probed with sera from RA (n = 30) and disease control samples (n = 30). Protein spots showing a sensitivity of >15% were identified by MS. 1‐D immunoblots revealed one protein band with a specificity in RA of 100%, a sensitivity of 43%, which was identified as fibrinogen β chain. 2‐D analysis revealed the subunits of fibrinogen, especially the β and γ chain, as the most prominent synovial autoantigens. We also identified vimentin, the Sa‐antigen and carbonic anhydrase I as a potentially new synovial autoantigen. The protein patterns of these immunoreactive spots were observed as trains. The spots showing the highest autoimmune reactivity occurred at the acidic side of these trains and were recognized by anticitrullinated protein/peptide antibodies positive RA sera. Antimodified citrulline staining of these patterns confirmed protein citrullination. Therefore, PTMs such as citrullination due to alterations of peptidylarginine deiminase activity or generation of RA‐specific epitopes, should be considered as a trigger in tolerance break.     

Characterization of a SAG‐InGaN/AlGaN LED by mixed‐source HVPE with a multi‐sliding boat system

Abstract— The selective area growth (SAG) of a InGaN/AlGaN light‐emitting diode (LED) is performed by using mixed‐source hydride vapor‐phase epitaxy (HVPE) with a multi‐sliding boat system. The SAG‐InGaN/AlGaN LED consists of a Si‐doped AlGaN cladding layer, an InGaN active layer, a Mg‐doped AlGaN cladding layer, and a Mg‐doped GaN capping layer. The carrier concentration of the n‐type Al_xGa_1?xN (x ～ 16%) cladding layer depends on the amount of poly‐Si placed in the Al‐Ga source. The carrier concentration is varied from 2.0 × 10¹⁶ to 1.1 × 10¹⁷ cm^?3. Electroluminescence (EL) characteristics show an emission peak wavelength at 426 nm with a full width at half‐maximum (FWHM) of approximately 0.47 eV at 20 mA. It was found that the mixed‐source HVPE method with a multi‐sliding boat system is a candidate growth method for III‐nitride LEDs.     

Protein carbonylation in kidney medulla of the spontaneously hypertensive rat

Enhanced generation of ROS has been reported in models of hypertension such as the spontaneously hypertensive rat (SHR). Impairment of kidney function has been implicated in development and progression of hypertension, and the renal medulla appears to play an important role in regulating long‐term blood pressure. A key biomarker of oxidative stress is the formation of protein carbonyls, which we set out to characterize in the SHR medulla. We identified 11 proteins that were differentially carbonylated in SHR medulla in comparison to normotensive wistars including enolase 1, catalase, carbonic anhydrase II, transferrin and members of the aldo–keto‐reductase family. This enhanced protein oxidation was not only accompanied by an increase in intracellular iron deposition, but aldo–keto‐reductase activity was also significantly less in SHR medulla than in normotensive Wistars. Oxidative stress appears selectively to target a subset of proteins in SHR kidney and modification of these proteins may in turn contribute to the renopathy associated with hypertension.     

The murine plasma protein response to polymicrobial intra-abdominal sepsis

Protein biomarkers in the peripheral blood could potentially be used as early indicators of sepsis and a means to stratify patients for clinical trials. Although individual molecular markers have been proposed for sepsis, none has clinical utility. The global changes in plasma proteins over the clinical course of sepsis have not been characterized using proteomic methods. We used cecal ligation and puncture to induce polymicrobial sepsis in mice and generated plasma protein profiles using 2‐D DIGE of plasma from septic mice and surgical controls. Replicate cohorts (n = 3) of 4–7 animals each were used to identify 62 gel features that changed significantly (Student's t‐test, p<0.05). We identified a suite of plasma proteins that describe uniquely the host plasma response to polymicrobial septic insult. Principal components analysis of protein abundance showed that ～90% of the variability between samples was due to sepsis. In addition to canonical acute phase proteins, we identified proteins that are associated with metabolic changes (e.g. α‐2 HS glycoprotein and zinc α‐2 glycoprotein) consistent with the pathophysiology of sepsis. The panel of sepsis‐associated molecular markers identified herein may prove useful in the diagnosis and categorization of sepsis.     

Toward the Proteome of the Human Peripheral Blood Eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom^® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10^?3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.     

Analysis of protein profiling studies of β‐thalassemia/Hb E disease

A number of studies have used global protein profiling technologies on a range of patient samples to detect proteins that are differentially expressed in β‐thalassemia/Hb E as an aid for understanding the physiopathology of this disease. Seven studies have identified a total of 111 unique, differentially expressed proteins. Seven proteins (prothrombin, alpha‐1‐antichymotrypsin, fibrinogen beta chain, hemoglobin beta, selenium‐binding protein, microtubule‐actin cross‐linking factor and adenomatous polyposis coli protein 2) have been identified in two independent studies, whereas two proteins (carbonic anhydrase 1 and peroxiredoxin‐2) have been identified in three independent studies. Both of these latter two proteins were consistently upregulated in the studies that identified them. Ontological analysis of all differentially regulated proteins identified “response to inorganic substances” as the most significant functional annotation cluster, which is consistent with iron overload being a major pathological consequence of this disease. Despite the range of samples investigated and the relatively small number of studies undertaken, a coherent picture of the mediators of the pathological consequences of β‐thalassemia/Hb E disease is starting to emerge.     

Proteomics integrated with Escherichia coli vector-based vaccines and antigen microarrays reveals the immunogenicity of a surface sialidase-like protein of Propionibacterium acnes

Proteomics is a powerful tool for the identification of proteins, which provides a basis for rational vaccine design. However, it is still a highly technical and time‐consuming task to examine a protein's immunogenicity utilizing traditional approaches. Here, we present a platform for effectively evaluating protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet‐c) was used as a representative antigen to establish this platform. A cell wall‐anchoring sialidase‐like protein (SLP) of Propionibacterium acnes was utilized to assess the efficacy of this platform. We constructed an Escherichia coli vector‐based vaccine by overexpressing Tet‐c or SLP in E. coli and utilized an intact particle of E. coli itself as a vaccine (E. coli Tet‐c or SLP vector). After ultraviolet (UV) irradiation, the E. coli vector‐based vaccines were administered intranasally into imprinting control region mice without adding exogenous adjuvants. For antibody detection, we fabricated antigen microarrays by printing with purified recombinant proteins including Tet‐c and SLP. Our results demonstrated that detectable antibodies were elicited in mice 6 weeks after intranasal administration of UV‐irradiated E. coli vector‐based vaccines. The antibody production of Tet‐c and SLP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the novel immunogenic SLP potentially serves as an antigen candidate for the development of vaccines targeting P. acnes‐associated diseases.     

Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays

Wegener's Granulomatosis (WG) is an idiopathic granulomatosis autoimmune vasculitis that primarily affects small vessels and is associated with glomerulonephritis and pulmonary granulomatous vasculitis. Anti‐neutrophil cytoplasmic auto‐antibodies (cANCA) against proteinase‐3 are used to identify WG, but ANCA titers are not present in some patients with the localized disease. The objective of this study was to develop an antibody array to help identify protein expression patterns in serum from patients with WG as compared to normals. The arrays were tested for limits of detection, background, and cross reactivity using standard proteins. The arrays were hybridized with either normal patient serum (n = 30) or with serum samples from a population of WG patients (n = 26) that were age and sex matched. Data analysis and curve fitting of the standard dilution series calculated r² values and determined a sensitivity of <50 pg/mL for the majority of proteins. A total of 24 proteins were assessed. Several statistically significant increases (p<0.05) were seen in the expression of: angiotensin converting enzyme‐I, IFN‐γ, IL‐8, s‐ICAM‐1 and s‐VCAM in WG patients as compared to controls. Utilizing the antibody microarray technology has led to the identification of potential biomarkers of vascular injury in the serum of WG patients.     

Interface and bulk effects for bias—light‐illumination instability in amorphous‐In—Ga—Zn—O thin‐film transistors

Abstract— Positive‐current‐bias (PB) instability and negative‐bias—light‐illumination (NBL) instability in amorphous‐In—Ga—Zn—O (a‐IGZO) thin‐film transistors (TFTs) have been examined. The channel‐ thickness dependence indicated that the Vth instability caused by the PB stress is primarily attributed to defects in the bulk a‐IGZO region for unannealed TFTs and to those in the channel—gate‐insulator interface for wet‐annealed TFTs. The interface and bulk defect densities (Dit and Nss, respectively) are Dit = 4.8 × 10¹¹ cm^?2/eV and Nss = 7.0×10¹⁶ cm^?3/eV for the unannealed TFT, which increased to 5.2×10¹¹ cm^?2/eV and 9.8×10¹⁶ cm^?3/eV, respectively, by the PB stress test. These are reduced significantly to Dit = 0.82×10¹¹ cm^?2/eV and Nss = 3.2×10¹⁶ cm^?3/eV for the wet‐annealed TFTs and are unchanged by the PB stress test. It was also found that the photo‐response of a‐IGZO TFTs begins at 2.3 eV of photon excitation, which corresponds to subgap states observed by photoemission spectroscopy. The origin of the NBL instability for the wet‐annealed TFTs is attributed to interface effects and considered to be a trap of holes at the channel‐gate—insulator interface where migration of the holes is enhanced by the electric field formed by the negative gate bias.     

Cooperative downconversion in Y3Al5O12 : RE3+,Yb3+ (RE = Tb,Tm, and Pr) nanophosphors

Abstract— Efficient near‐infrared (NIR) quantum cutting (QC) through cooperative downconversion in Y₃Al₅O₁₂ : RE³⁺,Yb³⁺ (RE = Tb, Tm, and Pr) nanophosphors has been demonstrated, which involves the conversion of the visible photon from RE³⁺ into the NIR emission of Yb³⁺ with the optimal quantum efficiency approaching 200%. The authors have analyzed the measured luminescence spectra and decay lifetimes and propose a mechanism to rationalize the NIR QC effect. The results indicate the potential of developing RE³⁺‐Yb³ dual‐ion‐based nanophosphors for the downconversion of high‐energy photons to multiple photons with an energy greater than the bandgap of silicon‐based‐photonics display devices.     

Very High Force Hydraulic McKibben Artificial Muscle with a p-Phenylene-2,6-benzobisoxazole Cord Sleeve

Small and lightweight actuators that generate high force and high energy are strongly required for realizing powerful robots and tools. By applying ultra-high-strength p-phenylene-2,6-benzobisoxazole fiber sleeves to McKibben artificial muscles, new hydraulic artificial muscles have been developed. While conventional McKibben muscles are driven by a maximum pneumatic pressure of 0.7 MPa, the newly developed muscles are driven by a maximum water hydraulic of pressure of 4 MPa, resulting in very high force capability. This paper presents the materials and structure of the new artificial muscle and the experimental results. The developed muscles are evaluated by four parameters — force density per volume (FDV), force density per mass (FDM), energy density per volume (EDV) and energy density per mass (EDM) — for comparisons with other conventional linear actuators. The prototype artificial muscle, which is 40 mm in diameter and 700 mm in length, can achieve a maximum contracting force of 28 kN, FDV of 32.3 × 10^–3 N/mm³, FDM of 9.44 × 10³ N/kg, EDV of 2600 × 10^–3 J/mm³ and EDM of 762 × 10³ J/kg. These values are 1.7 to 33 times larger than those of the typical conventional actuators. As the result, a high force artificial muscle of 40 mm in diameter that generates 28-kN contracting force has been developed successfully.     

Metal (IV) tetras (8‐hydroxyquinoline) (M = Zr,Hf) used as electroluminescent material and electron‐transport layer in OLEDs

Abstract— In this paper, we report on the utilization of zirconium (IV) tetras (8‐hydroxyquinoline), Zrq₄, and hafnium (IV) tetras (8‐hydroxyquinoline), Hfq₄, as an electroluminescent material in fluorescent organic light‐emitting diodes (OLED) and as electron transport layer (ETL) for high‐efficiency electrophosphorescent organic light‐emitting diodes (PHOLEDs). Structural studies show that the metal tetraquinolates (Mq₄) have a very low dipole moment (<0.1 D), in contrast to Alq₃ which has an estimated dipole moment of 4.7 D. Mobility measurements show that Mq₄ complexes give mobilities of (3.5 ± 0.5) × 10^?6 cm²/V‐sec, which are close to the values reported for Alq₃, i.e., (2.3–4.3) × 10^?6 cm²/V‐sec. OLEDs were prepared with the structure ITO/NPD (400 Å)/Mq_n (500 Å)/LiF/Al (NPD = 4‐4′‐bis[N‐(1‐naphthyl)‐N‐phenyl‐amino]bi phenyl, Mq_n = Alq₃, Zrq₄, Hfq₄. The Mq₄‐based OLEDs gave external efficiencies of 1.1%, while the Alq₃‐based devices of the same structure gave efficiencies of 0.7%. PHOLEDs have been fabricated with the structure ITO/NPD (500 Å)/CBP‐8% Ir(ppy)³ (250 Å)/BCP (150 Å)/Mq_n (250 Å)/LiF/Al (CBP = N,N′‐dicarbazolyl‐4‐4′‐biphenyl, Ir(ppy)³ = fac‐tris(2‐phenylpyrridine)iridium, BCP = bathocruprione). PHOLEDs with Mq₄ ETLs showed a greatly improved efficiency, when compared to Alq₃‐based PHOLEDs. The Zrq₄‐based PHOLEDs gave a peak external quantum efficiency of 14% at 0.3 mA/cm² (150 cd/m²), while the Hfq₄ based PHOLED gave a peak external quantum efficiency of 15% at 0.6 mA/cm² (300 cd/m²). Comparable PHOLEDs with an Alq₃ ETL give peak external quantum efficiencies of 8.0% at 0.5 mA/cm². The devices gave an electroluminescence (EL) spectrum consisting only of fac‐tris(2‐phenylpyrridine)iridium (Ir(ppy)³) dopant emission (CIE coordinates of 0.26, 0.66), with no Mq₄ emission observed at any bias level.     

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop. To discover sensitive and specific biomarkers for improvement of pre‐clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI‐TOF‐MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data. Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p<0.001) were identified as transthyretin (pre‐albumin) and C3a‐desArg by MS/MS and were further validated by antibody‐based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut‐off of <0.225 g/L for transthyretin and >1974 ng/mL for C3a‐desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.     

Two dimensional gel electrophoresis of apolipoprotein C‐III and MALDI‐TOF MS are complementary techniques for the study of combined defects in N‐ and mucin type O‐glycan biosynthesis

In the field of diseases related to glycosylation disorders, congenital defects associated with abnormalities in both O‐ and N‐glycosylation of proteins constitute arising novel entities. Defects in subunits of the conserved oligomeric Golgi protein complex have been shown to be involved in an important part of previously unsolved CDG type II combining abnormalities in both mucin type core1 O‐ and N‐glycans; furthermore, recent studies revealed that autosomal recessive cutis laxa type II could also be associated with such combined glycosylation defects. Based on the studies of serum samples from three patients including a case of cutis laxa, we present here evidence that 2‐DE of apolipoprotein C‐III in combination with MALDI‐TOF‐MS analysis of serum O‐ and N‐glycans allow the detection and the biochemical characterization of these newly recognized glycosylation disorders.     

Impacts of frequency and posture on body mass index in manual handling tasks

Workspace design plays an important role in ensuring workers' safety and welfare. The issue is more pressing in the manufacturing industry, where many workers must remain in a standing position, assume awkward postures, and perform repetitive tasks for extended periods of time. In this research, an electromyographic measurement technique was used to measure activity of four back muscles: the trapezius p. descendens, the deltoideus p. scapularis, the infraspinatus, and the latissimus dorsi. The results showed a statistically significant (p < 0.05) impact of left versus right side of the body, the degree of rotation to the right side of the body (0, 30, and 60 degrees rotation to the mid‐sagittal plane), body mass index level (normal weight [<25 kg/m²] vs. overweight [?25 kg/m²]), and tasks (high vs. low frequency). In this study, the role that workers and workspace variation play is clearly associated with an increase in the amplitude of electromyography at the targeted back muscles. © 2009 Wiley Periodicals, Inc.