Identification and characterization of protein cross-links induced by oxidative reactions

Introduction: Protein cross-links are common in biological systems and are generated both deliberately as a part of normal metabolism and also accidently as a result of exposure to external factors such as oxidation and glycation stresses. These cross-links can be both positive and negative for biological function, with high levels of inappropriate cross-links being associated with multiple human pathologies, as well as loss of protein structure and function in the food, agricultural, and pharmaceutical fields.

Areas covered: This review covers recent advances in the detection and identification of protein cross-links arising from oxidation reactions, mediated by both radicals and two-electron oxidants. Information on both enzymatic and nonenzymatic cross-linking is reviewed, both where this is intentional, as part of normal metabolism, and accidental and a potential cause of disease.

Expert commentary: The advantages and drawbacks of various methods available for the detection, identification, and quantification of these species are discussed, as well as some of the mechanisms and processes known to give rise to these species.     

Molecular basis of functional myogenic specification of Bona Fide multipotent adult cardiac stem cells

Ischemic Heart Disease (IHD) remains the developed world’s number one killer. The improved survival from Acute Myocardial Infarction (AMI) and the progressive aging of western population brought to an increased incidence of chronic Heart Failure (HF), which assumed epidemic proportions nowadays. Except for heart transplantation, all treatments for HF should be considered palliative because none of the current therapies can reverse myocardial degeneration responsible for HF syndrome. To stop the HF epidemic will ultimately require protocols to reduce the progressive cardiomyocyte (CM) loss and to foster their regeneration. It is now generally accepted that mammalian CMs renew throughout life. However, this endogenous regenerative reservoir is insufficient to repair the extensive damage produced by AMI/IHD while the source and degree of CM turnover remains strongly disputed. Independent groups have convincingly shown that the adult myocardium harbors bona-fide tissue specific cardiac stem cells (CSCs). Unfortunately, recent reports have challenged the identity and the endogenous myogenic capacity of the c-kit expressing CSCs. This has hampered progress and unless this conflict is settled, clinical tests of repair/regenerative protocols are unlikely to provide convincing answers about their clinical potential. Here we review recent data that have eventually clarified the specific phenotypic identity of true multipotent CSCs. These cells when coaxed by embryonic cardiac morphogens undergo a precisely orchestrated myogenic commitment process robustly generating bona-fide functional cardiomyocytes. These data should set the path for the revival of further investigation untangling the regenerative biology of adult CSCs to harness their potential for HF prevention and treatment.     

Vacuum Deposited Triple‐Cation Mixed‐Halide Perovskite Solar Cells

Hybrid lead halide perovskites are promising materials for future photovoltaics applications. Their spectral response can be readily tuned by controlling the halide composition, while their stability is strongly dependent on the film morphology and on the type of organic cation used. Mixed cation and mixed halide systems have led to the most efficient and stable perovskite solar cells reported, so far they are prepared exclusively by solution‐processing. This might be due to the technical difficulties associated with the vacuum deposition from multiple thermal sources, requiring a high level of control over the deposition rate of each precursor during the film formation. In this report, thermal vacuum deposition with multiple sources (3 and 4) is used to prepare for the first time, multications/anions perovskite compounds. These thin‐film absorbers are implemented into fully vacuum deposited solar cells using doped organic semiconductors. A maximum power conversion efficiency of 16% is obtained, with promising device stability. The importance of the control over the film morphology is highlighted, which differs substantially when these compounds are vacuum processed. Avenues to improve the morphology and hence the performance of fully vacuum processed multications/anions perovskite solar cells are proposed.     

Age at Introduction of Solid Food and Obesity Throughout the Life Course

Objective

This study aimed to examine the association between age at solid food (SF) introduction and obesity throughout the life course.

Methods

Among 31,816 mother– nurse daughter dyads in the Nurses' Mothers' Cohort Study and the Nurses' Health Study II, information was collected on age at SF introduction, body somatotype at ages 5 and 10, and Body Mass Index at age 18 and in adulthood. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for obesity throughout life were estimated using logistic regression models with adjustment for parental and nurse daughter covariates.

Results

Nurse daughters introduced to SF at ≥ 9 months versus 6 to 9 months had marginally higher age‐adjusted (OR: 1.21; 95% CI: 1.01, 1.47) and covariate‐adjusted (OR: 1.22; 95% CI: 1.01, 1.47) odds of obesity at age 5. Age at SF introduction was not related to obesity at ages 10 and 18 or in adulthood.

Conclusions

Late age at SF introduction was marginally associated with obesity at age 5, but this association did not persist throughout the life course.     

Effect of aging on mechanical stresses,deformations, and hemodynamics in human femoropopliteal artery due to limb flexion

Femoropopliteal artery (FPA) reconstructions are notorious for poor clinical outcomes. Mechanical and flow conditions that occur in the FPA with limb flexion are thought to play a significant role, but are poorly characterized. FPA deformations due to acute limb flexion were quantified using a human cadaver model and used to build a finite element model that simulated surrounding tissue forces associated with limb flexion-induced deformations. Strains and intramural principal mechanical stresses were determined for seven age groups. Computational fluid dynamics analysis was performed to assess hemodynamic variables. FPA shape, stresses, and hemodynamics significantly changed with age. Younger arteries assumed straighter positions in the flexed limb with less pronounced bends and more uniform stress distribution along the length of the artery. Even in the flexed limb posture, FPAs younger than 50 years of age experienced tension, while older FPAs experienced compression. Aging resulted in localization of principal mechanical stresses to the adductor hiatus and popliteal artery below the knee that are typically prone to developing vascular pathology. Maximum principal stresses in these areas increased threefold to fivefold with age with largest increase observed at the adductor hiatus. Atheroprotective wall shear stress reduced after 35 years of age, and atheroprone and oscillatory shear stresses increased after the age of 50. These data can help better understand FPA pathophysiology and can inform the design of targeted materials and devices for peripheral arterial disease treatments.     

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Background

The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50?kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

Methods

Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1?mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200?ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).

Results

The expected cDNA fragment of approximately 1240?bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10?mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.

Conclusions

The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

     

Methods for Physical Characterization of Phase-Separated Bodies and Membrane-less Organelles

Membrane-less organelles are cellular structures which arise through the phenomenon of phase separation. This process enables compartmentalization of specific sets of macromolecules (e.g., proteins, nucleic acids), thereby regulating cellular processes by increasing local concentration, and modulating the structure and dynamics of their constituents. Understanding the connection between structure, material properties and function of membrane-less organelles requires inter-disciplinary approaches, which address length and timescales that span several orders of magnitude (e.g., Ångstroms to micrometer, picoseconds to hours). In this review, we discuss the wide variety of methods that have been applied to characterize the morphology, rheology, structure and dynamics of membrane-less organelles and their components, in vitro and in live cells.     

Activation of the EPOR-β common receptor complex by cibinetide ameliorates impaired wound healing in mice with genetic diabetes

Diabetes is characterized by poor wound healing which currently lacks an efficacious treatment. The innate repair receptor (IRR) is a master regulator of tissue protection and repair which is expressed as a response injury or metabolic stress, including in diabetes. Activation of the IRR might provide benefit for diabetic wound healing. A specific IRR agonist cibinetide was administered in an incisional wound healing model performed mice with genetic diabetes (db⁺/db⁺) and compared to the normal wild-type. Animals were treated daily with cibinetide (30 μg/kg/s.c.) or vehicle and euthanized 3, 7, and 14 days after the injury to quantitate vascular endothelial growth factor (VEGF), malondialdehyde (MAL), phospho-Akt (pAkt), phospho e-NOS (p-eNOS), and nitrite/nitrate content within the wound. Additional evaluations included quantification of skin histological change, angiogenesis, scar strength, and time to complete wound closure. Throughout the wound healing process diabetic animals treated with vehicle exhibited increased wound MAL with reduced VEGF, pAkt, peNOS and nitrite/nitrate, all associated with poor re-epitheliziation, angiogenesis, and wound breaking strength. Cibenitide administration significantly improved these abnormalities. The results suggest that cibinetide-mediated IRR activation may represent an interesting strategy to treat diabetes-associated wound healing.     

Optimising the clinical strategy for autoimmune liver diseases: Principles of value-based medicine

Background

Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy.