Antibodies in proteomics II: screening,high-throughput characterization and downstream applications

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.     

Effect of Omacor on HRV parameters in patients with recent uncomplicated myocardial infarction – A randomized, parallel group, double-blind, placebo-controlled trial: study design [ISRCTN75358739]

Background

A large body of data derived from animal, epidemiological and clinical studies indicate that n-3 polyunsaturated fatty acids have a favourable effect on the prognosis of patients with cardiovascular disease in general, and on reducing sudden death in particular. Depressed heart rate variability (HRV), an indicator of impairment of the autonomic nervous system, has been shown to be a powerful predictor of subsequent mortality in patients surviving an acute myocardial infarction. A multitude of studies have demonstrated this strong association, suggesting that the imbalance in the sympathic/parasympathetic system may facilitate emergence of ventricular arrhythmias. Heart rate variability parameters will be assessed in the present study, with the primary objective of evaluating the possible superiority of Omacor (a highly refined, concentrated omega-3 fatty acid) versus placebo in improving HRV from baseline to endpoint in patients with recent uncomplicated myocardial infarction. Both groups will receive optimal conventional treatment. The study will also explore and quantify improvement in time domain HRV indices and will assess the safety of administering Omacor to optimally treated post-infarction patients (conventional treatment).

Methods

This multi-centre study will evaluate the effect of Omacor 1 g, o.d. on time-domain HRV parameters in comparison to placebo o.d. in patients with recent uncomplicated transmural myocardial infarction. Patients will be screened during the first few days after the acute event as appropriate for the patient's condition, and after obtaining informed consent. Based on inclusion/exclusion criteria, a first 24-hour Holter recording will be performed. Two to five days later, screened patients still eligible for the study will undergo a second 24-hour Holter recording. After the second Holter recording, all patients will be randomly allocated to treatment with Omacor 1 g, o.d. or placebo o.d. One hundred patients will be followed in double-blind fashion for a six-month period after randomization. Visits, including 24-hour Holter recording and assessment of adverse events, will take place at one-month intervals ± five days after randomization, i.e., six times in all.     

Archaeobotanical investigations in Liguria: preliminary data on the early Iron Age at Monte Trabocchetto (Pietra Ligure,Italy)

The archaeological site we studied is part of an early Iron Age hill fort (8th/7th cent. b.c.), located 800 m from the coast on the top of a hill named MonteTrabocchetto. This paper concerns an excavation, called saggio O, which disclosed a very varied stratigraphy characterised by highly anthropogenic layers and by a pit, presumably used as a silo for food storage, which was very rich in charred seeds and fruits. The study of the pit content showed the dominance of Hordeum vulgare, while Triticum dicoccon, T. monococcum, T. aestivum/durum, Panicum miliaceum and Setaria italica were less strongly represented. Some edible Leguminosae were also found (Lens culinaris, Vicia faba var. minor and V. ervilia). In the frequented areas around the pit, herbaceous weeds and fruit tree macro-remains were present (Prunus cf. spinosa, Corylus avellana, Quercus sp. and Vitis vinifera ssp. sylvestris). The identification of a large number of botanical taxa has provided important information on food of plant origin and agricultural practices during the early Iron Age on the Ligurian coast, the proto-historic archaeobotanical aspects of which are largely unknown.     

Enantiomerically pure tetrahydroquinoline derivatives as in vivo potent antagonists of the glycine binding site associated to the NMDA receptor

To identify neuroprotective agents after stroke, new substituted tetrahydroquinoline derivatives were designed as antagonists of the glycine binding site associated to the NMDA receptor, satisfying the key pharmacophoric requirements. In particular, the racemate 3c exhibited outstanding in vivo activity in the MCAo model in rats, when given iv both pre- and post-ischemia. Pure enantiomers 3c-(+) and 3c-(-) have been prepared following an original synthetic route. Despite the significant difference of activity observed in vitro, they shown similar neuroprotective profile in the MCAo model in rats.     

On the monodimensional approach to the estimation of the highest reynolds shear stress in a turbulent flow

The measurement of the Reynolds stress tensor, or at least of some of its components, is a necessary step to assess if the turbulence associated with the flow near prosthetic devices can damage blood constituents. Because of the intrinsic three dimensionality of turbulence, in general, a three-component anemometer should be used to measure directly the components of the Reynolds stress tensor. However, this can be practically unfeasible, especially in vivo; therefore, it is interesting to investigate the possibility of characterizing the turbulent flows that may occur in the circulatory system with the monodimensional data that a less complete equipment (e.g., a pulsed ultrasound Doppler) can yield. From the general expression of the Reynolds stress tensor, the highest shear stress can be deduced, as well as the Reynolds normal stress in the main flow direction. The relation between these two quantities, which is an issue already addressed in previous works, can thus be rigorously formulated in terms of some characteristic parameters of the Reynolds stress tensor, the principal normal stresses and the angles that the directions that define them form with the main flow direction. An experimental verification of the ratio of the two above-mentioned quantitites for the flow across bileaflet valves, investigated by means of two-dimensional laser Doppler anemometry, will illustrate the limitations of the monodimensional approach estimating the maximum load on blood constituents.     

Synthesis,biological evaluation,and SAR study of novel pyrazole analogues as inhibitors of Mycobacterium tuberculosis: Part 2. Synthesis of rigid pyrazolones

Two series of novel rigid pyrazolone derivatives were synthesized and evaluated as inhibitors of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis. Two of these compounds showed a high activity against MTB (MIC = 4 μg/mL). The newly synthesized pyrazolones were also computationally investigated to analyze if their properties fit the pharmacophoric model for antitubercular compounds previously built by us. The results are in agreement with those reported by us previously for a class of pyrazole analogues and confirm the fundamental role of the p-chlorophenyl moiety at C4 in the antimycobacterial activity.     

Prebiotic chemistry: chemical evolution of organics on the primitive Earth under simulated prebiotic conditions.

A series of prebiotic mixtures of simple molecules, sources of C, H, N, and O, were examined under conditions that may have prevailed during the Hadean eon (4.6-3.8 billion years), namely an oxygen-free atmosphere and a significant UV radiation flux over a large wavelength range due to the absence of an ozone layer. Mixtures contained a C source (methanol, acetone or other ketones), a N source (ammonia or methylamine), and an O source (water) at various molar ratios of C : H : N : O. When subjected to UV light or heated for periods of 7 to 45 days under an argon atmosphere, they yielded a narrow product distribution of a few principal compounds. Different initial conditions produced different distributions. The nature of the products was ascertained by gas chromatographic-mass spectral analysis (GC-MS). UVC irradiation of an aqueous methanol-ammonia-water prebiotic mixture for 14 days under low UV dose (6 x 10(-2) Einstein) produced methylisourea, hexamethylenetetramine (HMT), methyl-HMT and hydroxy-HMT, whereas under high UV dose (45 days; 1.9 x 10(-1) Einstein) yielded only HMT. By contrast, the prebiotic mixture composed of acetone-ammonia-water produced five principal species with acetamide as the major component; thermally the same mixture produced a different product distribution of four principal species. UVC irradiation of the CH(3)CN-NH(3)-H(2)O prebiotic mixture for 7 days gave mostly trimethyl-s-triazine, whereas in the presence of two metal oxides (TiO(2) or Fe(2)O(3)) also produced some HMT; the thermal process yielded only acetamide.     

Calpain-3 Impairs Cell Proliferation and Stimulates Oxidative Stress-Mediated Cell Death in Melanoma Cells

Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F₂-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F₂-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84^C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.     

Association of Individual Non-Steroidal Anti-Inflammatory Drugs and Chronic Kidney Disease: A Population-Based Case Control Study

Background

Non-steroidal anti-inflammatory agents (NSAIDs) are known to be associated with renal damage. No clear evidence exists regarding differential risk of chronic kidney disease (CKD), specifically, across various NSAIDs.

Aim

The aim of this population-based case-control study was to evaluate the association between use of individual NSAIDs and risk of CKD in a general population of Southern Italy.

Methods

A nested case-control study was carried out using the general practice Arianna database, identifying incident CKD patients as cases and matched controls from 2006 to 2011. The date of first CKD diagnosis was defined as the index date (ID). Conditional logistic regressions were performed to estimate the risk of CKD associated with NSAIDs by class and individual drugs as compared to non-use during different time windows (within one year, six or three months prior to ID), with the latter being defined as current users. Among current users, the effect of cumulative exposure to these drugs was evaluated.

Results

Overall, 1,989 CKD cases and 7,906 matched controls were identified. A statistically significant increase in the risk of CKD was found for current users of oxicams (adjusted OR: 1.68; 95% CI: 1.15-2.44) and concerning individual compounds, for ketorolac (adj. OR: 2.54; 95% CI: 1.45-4.44), meloxicam (adj. OR: 1.98; 95% CI: 1.01-3.87) and piroxicam (adj. OR: 1.95; 95% CI: 1.19-3.21).

Conclusions

The risk of CKD varies across individual NSAIDs. Increased risk has been found for ketorolac, which may precipitate subclinical CKD through acute renal damage, and long-term exposure to oxicams, especially meloxicam and piroxicam.     

Plasma Free Hemoglobin and Microcirculatory Response to Fresh or Old Blood Transfusions in Sepsis

BackgroundFree hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected.MethodsThis is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO₂) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed.ResultsSimilar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003).ConclusionsOld RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01584999","term_id":"NCT01584999"}}NCT01584999