Engineering of the Dsb (disulfide bond) proteins – contribution towards understanding their mechanism of action and their applications in biotechnology and medicine

Abstract

The Dsb protein family in prokaryotes catalyzes the generation of disulfide bonds between thiol groups of cysteine residues in nascent proteins, ensuring their proper three-dimensional structure; these bonds are crucial for protein stability and function. The first Dsb protein, Escherichia coli DsbA, was described in 1991. Since then, many details of the bond-formation process have been described through microbiological, biochemical, biophysical and bioinformatics strategies. Research with the model microorganism E. coli and many other bacterial species revealed an enormous diversity of bond-formation mechanisms. Research using Dsb protein engineering has significantly helped to reveal details of the disulfide bond formation. The first part of this review presents the research that led to understanding the mechanism of action of DsbA proteins, which directly transfer their own disulfide into target proteins. The second part concentrates on the mechanism of electron transport through the cell cytoplasmic membrane. Third and lastly, the review discusses the contribution of this research towards new antibacterial agents.     

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes)

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585–1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96–95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.     

Structural evolution of water and hydroxyl groups during thermal,mechanical and chemical treatment of high purity natural quartz

Fused silica crucibles are commonly used in the fabrication process of solar grade silicon ingots. These crucibles are manufactured from high purity natural quartz sand and as a consequence, their properties are influenced by the presence of water and hydroxyls in the raw quartz. In this work, diffuse reflectance IR, ¹H magic angle spinning NMR, and Raman spectroscopy were used to investigate the influence of thermal treatment on water and hydroxyl groups in high purity natural quartz sand. Most of the water in dry sand is present in the form of closed inclusions within the quartz grains which were detected in Raman imaging studies, even after thermally treating the samples at 600 °C. Only after heating to 900 °C did this water completely vanish, most likely as a result of rupturing of the inclusions. However, newly formed OH groups, identified as isolated and hydrogen bound OH were observed as products of the reaction between water and quartz. Similarly, liquid water was observed in NMR spectra even after treatment at 600 °C while at temperatures >900 °C, only non-interacting silanol groups were present. The comparison of the temperature dependence of the IR and NMR spectra also yields insight into the assignment of the OH stretching mode region of the IR spectrum in this system. The intensity of water related bands decreases while the intensity of OH bands first increases and then decreases with increasing temperature. The band intensity of Al-rich defects as well as the characteristic feature at 3200 cm⁻¹ does not follow the temperature dependence of typical water peaks. It is also shown that leaching the quartz sand in HF solution helps to remove water from inclusions, likely by forming pathways for fluid flow inside the quartz grains. Milling of the samples caused formation of an additional type of hydroxyl group, possibly due to partial amorphisation of the surfaces of the quartz grains surface during the process. The results improve the basis for a knowledge-based processes development for the processing of high purity natural quartz.

In dry quartz stable closed liquid micron-size inclusions and newly formed OH groups were observed after thermal treatment.     

Preliminary studies on the cestodofauna of goosander Mergus merganser L., 1758 from West Pomerania

The research included 54 individuals of the goosander Mergus merganser L., 1758 (Anseriformes, Mergini), wintering on Lake Dabie within the administrative limits of Szczecin. Seven species of cestoda from two families were found in those ducks. Two species from the family Diphyllobothriidae were recorded: Ligula intestinalis (Linnaeus, 1758) and Schistocephalus solidus (Müller, 1776), and five species from family Hymenolepididae: Dicranotaenia mergi Yamaguti, 1940, Fimbriaria mergi Grytner-Ziecina et Cielecka, 1995, Microsomacanthus vistulae (Czaplinski, 1960), Retinometra macracanthos (von Linstow, 1877) oraz Tschertkovilepis tenuirostris (Rudolphi, 1819). Founding species Dicranotaenia mergi is the first observation of this type in Poland.     

Periscope for noninvasive two-photon imaging of murine retina in vivo

Two-photon microscopy allows visualization of subcellular structures in the living animal retina. In previously reported experiments it was necessary to apply a contact lens to each subject. Extending this technology to larger animals would require fitting a custom contact lens to each animal and cumbersome placement of the living animal head on microscope stage. Here we demonstrate a new device, periscope, for coupling light energy into mouse eye and capturing emitted fluorescence. Using this periscope we obtained images of the RPE and their subcellular organelles, retinosomes, with larger field of view than previously reported. This periscope provides an interface with a commercial microscope, does not require contact lens and its design could be modified to image retina in larger animals.OCIS codes: (170.0110) Imaging systems, (170.4460) Ophthalmic optics and devices, (170.5755) Retina scanning, (180.4315) Nonlinear microscopy, (330.7327) Visual optics, ophthalmic instrumentation     

Renal Mechanisms of Association between Fibroblast Growth Factor 1 and Blood Pressure

The fibroblast growth factor 1 (FGF1) gene is expressed primarily in the kidney and may contribute to hypertension. However, the biologic mechanisms underlying the association between FGF1 and BP regulation remain unknown. We report that the major allele of FGF1 single nucleotide polymorphism rs152524 was associated in a dose-dependent manner with systolic BP (P=9.65×10⁻⁵) and diastolic BP (P=7.61×10⁻³) in a meta-analysis of 14,364 individuals and with renal expression of FGF1 mRNA in 126 human kidneys (P=9.0×10⁻³). Next-generation RNA sequencing revealed that upregulated renal expression of FGF1 or of each of the three FGF1 mRNA isoforms individually was associated with higher BP. FGF1-stratified coexpression analysis in two separate collections of human kidneys identified 126 FGF1 partner mRNAs, of which 71 and 63 showed at least nominal association with systolic and diastolic BP, respectively. Of those mRNAs, seven mRNAs in five genes (MME, PTPRO, REN, SLC12A3, and WNK1) had strong prior annotation to BP or hypertension. MME, which encodes an enzyme that degrades circulating natriuretic peptides, showed the strongest differential coexpression with FGF1 between hypertensive and normotensive kidneys. Furthermore, higher level of renal FGF1 expression was associated with lower circulating levels of atrial and brain natriuretic peptides. These findings indicate that FGF1 expression in the kidney is at least under partial genetic control and that renal expression of several FGF1 partner genes involved in the natriuretic peptide catabolism pathway, renin-angiotensin cascade, and sodium handling network may explain the association between FGF1 and BP.     

Interleukin 6 -174(G>C) gene polymorphism is related to celiac disease and autoimmune thyroiditis coincidence in diabetes type 1 children

The aim of the study was to assess the relationship between IL-6 gene polymorphism at -174(G>C) and the coincidence of celiac and autoimmune thyroid diseases with type 1 diabetes mellitus (DM1) in children. 200 children with DM1 aged 13.23+/-3.54 years and 172 healthy controls were analyzed. The IL-6 gene -174(G>C) polymorphism at the promoter region of the gene was analyzed by the PCR-RFLP method. The genotype distribution was significantly different in diabetic children as compared to the healthy controls (p=0.01). In DM1 patients GC heterozygotes were the most common (52.5%), while CC homozygotes accuted for 29% and GG homozygotes only for 18% of cases. In contrast, GG homozygotes were much more frequent among healthy children (31%). Besides, the GG homozygotes were significantly more frequent among diabetic children with celiac disease (p=0.04) in relation to those without autoimmune complications. In children with autoimmune thyroiditis, the distribution of the IL-6 genotypes was similar to that seen in diabetic patients without autoimmune complications (p=0.24). The results of our study suggest that the diabetic children, who have IL-6 gene -174GG genotype may have an increased risk for celiac disease development.