High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

Background: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.Pubblicat

The Environmental Effects of the Innovative Ejectors Plant Technology for the Eco-Friendly Sediment Management in Harbors

A sediment bypassing plant based on innovative jet pump, ejectors, has been tested in the first-of-a-kind demo application at the harbor of Cervia (Italy, Northern Adriatic Sea). The ejector is a jet pump aimed to reduce sediment accumulation in navigation channels and coastal areas. Herein we present results of the first study assessing the potential ecological effects of the ejectors plant. Sediment characteristics, benthic, and fish assemblages before and after the plant activation have been analyzed in the putatively impacted (the sediment removal and discharge) areas and four control locations, one time before and two times after plant activation. Ejectors plant operation resulted in a reduction of the mud and organic matter content in the sediment, as well as in changes in shell debris amount in the impacted areas. Abundance and species richness of benthic macroinvertebrates, initially reduced in the impacted areas, probably due to the previous repeated dredging, returned to higher values during demo plant continuous operation. Higher diversity of fish fauna was observed in the study area during plant operation period. Observed dynamics of the ecological status of the marine habitat suggest that an ejectors plant could represent an eco-friendly solution alternative to dredging operations to solve harbor siltation problems

Expression of CD4 on human peripheral blood neutrophils

AbstractCD4, the primary receptor for entry of HIV, is known to be expressed on T cells and monocytes/macrophages; healthy natural killer (NK) lymphocytes; in vitro human herpesvirus 6 (HHV6)–infected CD8+, NK, and γδ T lymphocytes; CD34+ progenitor cells; and a subset of eosinophils and basophils. We here report the unconventional expression of CD4 at the surface of peripheral blood neutrophils derived from 4 of 51 (7.8%) HIV-1–infected and 3 of 25 (12%) uninfected donors, with similar frequency within the 2 groups. The percentage of CD4+ neutrophils ranged from 39% to 97% of the total neutrophil population. Both surface and cytoplasmic forms of CD4 were present in neutrophils. Quantitative RNA polymerase chain reaction (PCR) revealed that neutrophils contain levels of CD4 mRNA comparable to those of peripheral blood mononuclear cells derived from the same donor. The conformation of CD4 expressed at the surface of neutrophils was similar to that of CD4 expressed on T lymphocytes as determined by the binding of monoclonal antibodies specific for conformational epitopes and the binding of recombinant HIV-1 gp120. Thus, our data provide evidence that neutrophils express endogenous CD4 and bind HIV. Owing to their abundance in peripheral blood, CD4+ neutrophils may influence significantly the biodistribution of HIV delivering it to sites of inflammation or to additional tissue reservoirs

Candidate gene study of HOXB1 in autism spectrum disorder

<p>Abstract</p> <p>Background</p> <p><it>HOXB1 </it>plays a major role in brainstem morphogenesis and could partly determine the cranial circumference in conjunction with <it>HOXA1</it>. In our sample, <it>HOXA1 </it>alleles significantly influence head growth rates both in autistic patients and in population controls. An initial report, suggesting that <it>HOXB1 </it>could confer autism vulnerability in interaction with <it>HOXA1</it>, was not confirmed by five small association studies.</p> <p>Methods</p> <p>Our sample includes 269 autistic individuals, belonging to 219 simplex and 28 multiplex families. A mutational analysis of the two exons and flanking intronic sequences of the <it>HOXB1 </it>gene was carried out in 84 autistic patients by denaturing high performance liquid chromatography, followed by DNA sequencing. Identified rare variants were then searched by a restriction analysis in 236 autistic patients and 325-345 controls. Case-control and family-based association studies were performed on two common variants in 169 Italian patients versus 184 Italian controls and in 247 trios.</p> <p>Results</p> <p>We identified three common polymorphisms, rs72338773 [c.82insACAGCGCCC (INS/nINS)], rs12939811 [c.309A>T (Q103H)], and rs7207109 [c.450G>A (A150A)] and three rare variants, namely IVS1+63G>A, rs35115415 [c.702G>A (V234V)] and c.872_873delinsAA (S291N). SNPs rs72338773 and rs12939811 were not associated with autism, using either a case-control (alleles, exact <it>P </it>= 0.13) or a family-based design [transmission/disequilibrium test (TDT)χ²= 1.774, <it>P </it>= 0.183]. The rare variants, all inherited from one of the parents, were present in two Italian and in two Caucasian-American families. Autistic probands in two families surprisingly inherited a distinct rare variant from each parent. The IVS1+63A allele was present in 3/690 control chromosomes, whereas rare alleles at rs35115415 and c.872_873delinsAA (S291N) were not found in 662 and 650 control chromosomes, respectively. The INS-T309 allele influenced head size, but its effect appears more modest and shows no interaction with <it>HOXA1 </it>alleles. The INS-T309 allele is also associated with more severe stereotypic behaviours, according to ADI-R scores (<it>N </it>= 60 patients, <it>P </it>< 0.01).</p> <p>Conclusions</p> <p><it>HOXB1 </it>mutations do not represent a common cause of autism, nor do <it>HOXB1 </it>common variants play important roles in autism vulnerability. <it>HOXB1 </it>provides minor, albeit detectable contributions to head circumference in autistic patients, with <it>HOXA1 </it>displaying more prominent effects. <it>HOXB1 </it>variants may modulate the clinical phenotype, especially in the area of stereotypic behaviours.</p

Superparamagnetic Nanoparticles as High Efficiency Magnetic Resonance Imaging T-2 Contrast Agent

Nanoparticle-based magnetic resonance imaging T-2 negative agents are of great interest, and much effort is devoted to increasing cell loading capability while maintaining low cytotoxicity. Herein, two classes of mixed-ligand protected magnetic-responsive, bimetallic gold/iron nano particles (Au/Fe NPs) synthesized by a two-step method are presented. Their structure, surface composition, and magnetic properties are characterized. The two classes of sulfonated Au/Fe NPs, with an average diameter of 4 nm, have an average atomic ratio of Au to Fe equal to 7 or 8, which enables the Au/Fe NPs to be superparamagnetic with a blocking temperature of 56 K and 96 K. Furthermore, preliminary cellular studies reveal that both Au/Fe NPs show very limited toxicity. MRI phantom experiments show that r(2)/r(1) ratio of Au/Fe NPs is as high as 670, leading to a 66% reduction in T-2 relaxation time. These nanoparticles provide great versatility and potential for nanopartide-based diagnostics and therapeutic applications and as imaging contrast agents

The Environmental Effects of the Innovative Ejectors Plant Technology for the Eco-Friendly Sediment Management in Harbors

A sediment bypassing plant based on innovative jet pump, ejectors, has been tested in the first-of-a-kind demo application at the harbor of Cervia (Italy, Northern Adriatic Sea). The ejector is a jet pump aimed to reduce sediment accumulation in navigation channels and coastal areas. Herein we present results of the first study assessing the potential ecological effects of the ejectors plant. Sediment characteristics, benthic, and fish assemblages before and after the plant activation have been analyzed in the putatively impacted (the sediment removal and discharge) areas and four control locations, one time before and two times after plant activation. Ejectors plant operation resulted in a reduction of the mud and organic matter content in the sediment, as well as in changes in shell debris amount in the impacted areas. Abundance and species richness of benthic macroinvertebrates, initially reduced in the impacted areas, probably due to the previous repeated dredging, returned to higher values during demo plant continuous operation. Higher diversity of fish fauna was observed in the study area during plant operation period. Observed dynamics of the ecological status of the marine habitat suggest that an ejectors plant could represent an eco-friendly solution alternative to dredging operations to solve harbor siltation problems