Configuration and optimization of a common two-site immunoassay for human prolactin using a chemiluminescent tracer and an enzymatic tracer

We have developed a two-site immunoassay for human prolactin using two monoclonal antibodies: one of them immobilized on the solid phase and the other labelled with biotin. The serum is incubated simultaneously with the antibody-coated bead, the biotinylated antibody and the tracer (streptavidin–isoluminol or avidin–peroxidase complex). Our experimental work has been directed towards a common set of reagents to capture the prolactin and then, with different tracers, towards obtaining on the calibration curve the same results for unknown samples. On the basis of the positive results we obtained, we have developed a kit that can be used by the customer or as an enzyme-immunoassay or as a chemiluminescent immunoassay, depending on instrumentation available, spectrophotometer or luminometer.     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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Biomechanical analysis of pedalling for rehabilitation purposes: experimental results on two pathological subjects and comparison with non-pathological findings

In this paper the experimental results obtained by means of a prototype measuring device dedicated to the evaluation of the rehabilitation level of the lower limb are presented. The analysis of the experimental data collected on non-pathological subjects allows the identification of the characteristic meaning of the most significant parameters typical of healthy subjects. These data have been employed for a systematic comparison with the same parameters measured on two pathological subjects, in order to define quantitative indicators of the rehabilitation degree of the lower limbs and indicators of the "quality" of the movement.     

The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant

Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO.     

Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.     

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein ��2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.     

Biomechanical Analysis of Pedalling for Rehabilitation Purposes: Experimental Results on Two Pathological Subjects and Comparison with Non-pathological Findings

In this paper the experimental results obtained by means of a prototype measuring device dedicated to the evaluation of the rehabilitation level of the lower limb are presented. The analysis of the experimental data collected on non-pathological subjects allows the identification of the characteristic meaning of the most significant parameters typical of healthy subjects. These data have been employed for a systematic comparison with the same parameters measured on two pathological subjects, in order to define quantitative indicators of the rehabilitation degree of the lower limbs and indicators of the “quality” of the movement.     

Hospitalization Rates and Post-Operative Mortality for Abdominal Aortic Aneurysm in Italy over the Period 2000–2011

Background

Recent studies have reported declines in incidence, prevalence and mortality for abdominal aortic aneurysms (AAAs) in various countries, but evidence from Mediterranean countries is lacking. The aim of this study is to examine the trend of hospitalization and post-operative mortality rates for AAAs in Italy during the period 2000–2011, taking into account the introduction of endovascular aneurysm repair (EVAR) in 1990s.

Methods

This retrospective cohort study was carried out in Emilia-Romagna, an Italian region with 4.5 million inhabitants. A total of 19,673 patients hospitalized for AAAs between 2000 and 2011, were identified from the hospital discharge records (HDR) database. Hospitalization rates, percentage of OSR and EVAR and 30-day mortality rates were calculated for unruptured (uAAAs) and ruptured AAAs (rAAAs).

Results

Adjusted hospitalization rates decreased on average by 2.9% per year for uAAAs and 3.2% for rAAAs (p<0.001). The temporal trend of 30-day mortality rates remained stable for both groups. The percentage of EVAR for uAAAs increased significantly from 2006 to 2011 (42.7 versus 60.9% respectively, mean change of 3.9% per year, p<0.001). No significant difference in mortality was found between OSR and EVAR for uAAAs and rAAAs.

Conclusions

The incidence and trend of hospitalization rates for rAAAs and uAAAs decreased significantly in the last decade, while 30-day mortality rates in operated patients remained stable. OSR continued to be the most common surgery in rAAAs, although the gap between OSR and EVAR recently declined. The EVAR technique became the preferred surgery for uAAAs since 2008.     

Application of a semi-automatic cartilage segmentation method for biomechanical modeling of the knee joint

Manual segmentation of articular cartilage from knee joint 3D magnetic resonance images (MRI) is a time consuming and laborious task. Thus, automatic methods are needed for faster and reproducible segmentations. In the present study, we developed a semi-automatic segmentation method based on radial intensity profiles to generate 3D geometries of knee joint cartilage which were then used in computational biomechanical models of the knee joint. Six healthy volunteers were imaged with a 3T MRI device and their knee cartilages were segmented both manually and semi-automatically. The values of cartilage thicknesses and volumes produced by these two methods were compared. Furthermore, the influences of possible geometrical differences on cartilage stresses and strains in the knee were evaluated with finite element modeling. The semi-automatic segmentation and 3D geometry construction of one knee joint (menisci, femoral and tibial cartilages) was approximately two times faster than with manual segmentation. Differences in cartilage thicknesses, volumes, contact pressures, stresses, and strains between segmentation methods in femoral and tibial cartilage were mostly insignificant (p > 0.05) and random, i.e. there were no systematic differences between the methods. In conclusion, the devised semi-automatic segmentation method is a quick and accurate way to determine cartilage geometries; it may become a valuable tool for biomechanical modeling applications with large patient groups.