Shotgun phage display — Selection for bacterial receptins or other exported proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins. Published: May 1, 2003     

Microwave thermotherapy of prostatic enlargement--analysis of radiometric thermometry using a hybrid bio-heat equation

The radiometric temperature measurement included in a commercial device for transurethral microwave thermotherapy (TUMT) of the prostate was investigated utilizing both phantom experiments and computer simulations. Two finite element (FE) models were developed. One is in part based on the experimental results, and serves as a complement to the experiments, while the other describes a perfused tissue situation for which the hybrid bio-heat equation was used to model the thermal effects of blood perfusion. The aim of the study was to investigate how the radiometric thermometer is affected by the temperature close to the antenna, and to analyze the relation between blood perfusion, temperature distribution and radiometric temperature measurement. It was found that the radiometric temperature was affected to a greater extent by the temperature very close to the antenna, in contrast to what has been expected in previous studies. The blood perfusion was found to mainly affect the temperature distribution outside the maximum temperature (located 2-3 mm outside the cooled catheter). Thus, the relation between the radiometric temperature and the temperature in the treated area is relatively weak.     

Comprehensive proteome analysis of nasal lavage samples after controlled exposure to welding nanoparticles shows an induced acute phase and a nuclear receptor,LXR/RXR,activation that influence the status of the extracellular matrix

Background

Epidemiological studies have shown that many welders experience respiratory symptoms. During the welding process a large number of airborne nanosized particles are generated, which might be inhaled and deposited in the respiratory tract. Knowledge of the underlying mechanisms behind observed symptoms is still partly lacking, although inflammation is suggested to play a central role. The aim of this study was to investigate the effects of welding fume particle exposure on the proteome expression level in welders suffering from respiratory symptoms, and changes in protein mediators in nasal lavage samples were analyzed. Such mediators will be helpful to clarify the pathomechanisms behind welding fume particle-induced effects.

Methods

In an exposure chamber, 11 welders with work-related symptoms in the lower airways during the last month were exposed to mild-steel welding fume particles (1 mg/m³) and to filtered air, respectively, in a double-blind manner. Nasal lavage samples were collected before, immediately after, and the day after exposure. The proteins in the nasal lavage were analyzed with two different mass spectrometry approaches, label-free discovery shotgun LC–MS/MS and a targeted selected reaction monitoring LC–MS/MS analyzing 130 proteins and four in vivo peptide degradation products.

Results

The analysis revealed 30 significantly changed proteins that were associated with two main pathways; activation of acute phase response signaling and activation of LXR/RXR, which is a nuclear receptor family involved in lipid signaling. Connective tissue proteins and proteins controlling the degradation of such tissues, including two different matrix metalloprotease proteins, MMP8 and MMP9, were among the significantly changed enzymes and were identified as important key players in the pathways.

Conclusion

Exposure to mild-steel welding fume particles causes measurable changes on the proteome level in nasal lavage matrix in exposed welders, although no clinical symptoms were manifested. The results suggested that the exposure causes an immediate effect on the proteome level involving acute phase proteins and mediators regulating lipid signaling. Proteases involved in maintaining the balance between the formation and degradation of extracellular matrix proteins are important key proteins in the induced effects.

     

The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infections

Dramatic increase in bacterial resistance towards conventional antibiotics emphasises the importance to identify novel, more potent antimicrobial therapies. Antimicrobial peptides (AMPs) have emerged as a promising new group to be evaluated in therapeutic intervention of infectious diseases. Here we describe a novel AMP, PXL150, which demonstrates in vitro a broad spectrum microbicidal action against both Gram-positive and Gram-negative bacteria, including resistant strains. The potent microbicidal activity and broad antibacterial spectrum of PXL150 were not associated with any hemolytic activity. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance towards PXL150 during continued selection pressure. PXL150 caused a rapid depolarisation of cytoplasmic membrane of S. aureus, and dissipating membrane potential is likely one mechanism for PXL150 to kill its target bacteria. Studies in human cell lines indicated that PXL150 has anti-inflammatory properties, which might be of additional benefit. PXL150 demonstrated pronounced anti-infectious effect in an in vivo model of full thickness wounds infected with MRSA in rats and in an ex vivo model of pig skin infected with S. aureus. Subcutaneous or topical application of the peptide in rats did not lead to any adverse reactions. In conclusion, PXL150 may constitute a new therapeutic alternative for local treatment of infections, and further studies are warranted to evaluate the applicability of this AMP in clinical settings.     

The impact of temperature on the metabolome and endocrine metabolic signals in Atlantic salmon (Salmo salar)

The aim was to elucidate the effects of elevated temperature on growth performance, growth- and appetite-regulating hormones and metabolism in Atlantic salmon, Salmo salar. Post-smolts in seawater (average mass 175 g) that had been reared at 12 °C were kept at three temperatures (8, 12 and 18 °C) and sampled after one and three months. After three months, the fish kept in 18 °C had decreased growth rate and condition factor, and elevated plasma levels of growth hormone (GH) and leptin, compared with fish kept at the lower temperatures. Food conversion efficiency was also decreased at 18 °C, while at the same time protein uptake was improved and thus was not a limiting mechanism for growth. Redistribution of energy stores in fish at the highest temperature is evident as a preference of maintaining length growth during times of limited energy availability. NMR-based metabolomics analyses of plasma revealed that several metabolites involved in energy metabolism were negatively affected by temperature in the upper temperature range of Atlantic salmon. Specifically, the high temperature induced a decline of several amino acids (glutamine, tyrosine and phenylalanine) and a shift in lipid metabolism. It appears likely that the decreased food intake at the highest temperature is linked to an anorexigenic function of leptin, but also that the decreased food intake, feed conversion efficiency and condition factor can be linked to changes in GH endocrinology.     

Norepinephrine transporter heterozygous knockout mice exhibit altered transport and behavior

The norepinephrine (NE) transporter (NET) regulates synaptic NE availability for noradrenergic signaling in the brain and sympathetic nervous system. Although genetic variation leading to a loss of NET expression has been implicated in psychiatric and cardiovascular disorders, complete NET deficiency has not been found in people, limiting the utility of NET knockout mice as a model for genetically driven NET dysfunction. Here, we investigate NET expression in NET heterozygous knockout male mice (NET^+/?), demonstrating that they display an approximately 50% reduction in NET protein levels. Surprisingly, these mice display no significant deficit in NET activity assessed in hippocampal and cortical synaptosomes. We found that this compensation in NET activity was due to enhanced activity of surface‐resident transporters, as opposed to surface recruitment of NET protein or compensation through other transport mechanisms, including serotonin, dopamine or organic cation transporters. We hypothesize that loss of NET protein in the NET^+/? mouse establishes an activated state of existing surface NET proteins. The NET^+/? mice exhibit increased anxiety in the open field and light–dark box and display deficits in reversal learning in the Morris water maze. These data suggest that recovery of near basal activity in NET^+/? mice appears to be insufficient to limit anxiety responses or support cognitive performance that might involve noradrenergic neurotransmission. The NET^+/? mice represent a unique model to study the loss and resultant compensatory changes in NET that may be relevant to behavior and physiology in human NET deficiency disorders .