DESIGN OF A HIGH-THROUGHPUT METHODFOR THE ASSESSMENT OF ENZYME ACTIVITY UPON ADSORPTION ONCLUSTER-ASSEMBLED NANOSTRUCTURED TITANIUM OXIDE FILMS

Control of nanometre-scale topography of solid surfaces has opened the possibility to tailor the interactions between materials and biomolecules maintaining the biological functions of these molecules, a crucial aspect for diverse biomaterial applications. However, the primary mechanisms that dictate protein adsorption to topographical nanostructures are often poorly understood. We have addressed this question by scrutinizing the catalytic activity of immobilized serine-protease trypsin as product of its adsorption properties on cluster-assembled nanostructured titania films. Both, adsorption and activity of surface-bound enzymes were evaluated in parallel using innovative microarray-based methodology developed in this PhD work. Trypsin adsorption analysis demonstrated an increment with roughness of Langmuir parameters \u2013 saturation uptake and equilibrium dissociation constant, that exceeded the contribution predicted by increase in sample specific area. This finding was interpreted by the clustering of protein molecules inside titania surface nanopores, a model proposed in our previous study of three non-enzymatic proteins. The growth of adsorbed trypsin activity with roughness was attributed to the increase in specific area of titania films, whereas the drop in specific activity resulted from steric hindrance of trypsin clustered inside titania nanopores. This study has shed light on the topographical determinants of trypsin adsorption on nanostructured titania surfaces and its impact on trypsin activity. A novel method was developed elucidating the obstacles and specifics of protease immobilization by physisorption and suggesting possible routes to solve them. This methodology is directly applicable in biomaterial screening with respect to the functionality of immobilized enzymes and can be extended beyond the trypsin-nanostructured titania model

Association of variants in the CP, ATOX1 and COMMD1 genes with Wilson disease symptoms in Latvia

Funding Information: This study was partially financed by a grant of Riga Stradins University, Department of Doctoral studies and grant of Roche Academy. Funding Information: Funding . This study was partially financed by a grant of Riga Stradiņš University, Department of Doctoral studies and grant of Roche Academy. Publisher Copyright: © 2019 Zarina A, Tolmane I, Krumina Z, Tutane AI, Gailite L, published by Sciendo.Wilson's disease (WD) is a copper metabolism disorder, caused by allelic variants in the ATP7B gene. Wilson's disease can be diagnosed by clinical symptoms, increased copper and decreased cerulopasmin levels, which could all also be by other genetic variants beyond the ATP7B gene, e.g., disturbed ceruloplasmin biosynthesis can be caused by pathogenic allelic variants of the CP gene. Copper metabolism in the organism is affected by several molecules, but pathogenic variants and related phenotypes are described with COMMD1 and ATOX1 genes. The aim of the study was to test other genes, CP, ATOX1 and COMMD1, for possible influence to the manifestation of WD. Patients were enrolled on the basis of Leipzig's diagnostic criteria, 64 unrelated patients with confirmed WD. Direct sequencing of promoter region of the CP gene and ATOX1 and COMMD1 gene exons was conducted. Statistically significant differences were found between the two variants in the CP gene and the ATP7B genotype (rs66508328 variant AA genotype and the rs11708215 variant GG genotype) were more common in WD patients with an unconfirmed ATP7B genotype. One allelic (intronic) variant was found in the ATOX1 gene without causing the functional changes of the gene. Three allelic variants were identified in the COMMD1 gene. No statistically significant differences were found between allele and genotype frequencies and the first clinical manifestations of WD. Different variants of the CP gene contributed to a WD-like phenotype in clinically confirmed WD patients with neurological symptoms and without identified pathogenic variants in the ATP7B gene. Allelic variants in the ATOX1 and COMMD1 genes do not modify the clinical manifestation of WD in Latvian patients.publishersversionPeer reviewe

Clinical and Genetic Characterisation of Cystic Fibrosis Patients in Latvia : A Twenty-Five-Year Experience

publishersversionPeer reviewe

Plasma neurofilament light chain as a potential biomarker in Charcot‐Marie‐Tooth disease

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a chronic, slowly progressing disorder. The lack of specific disease progression biomarkers limits the execution of clinical trials. However, neurofilament light chain (NfL) has been suggested as a potential biomarker for peripheral nervous system disorders. METHODS: Ninety-six CMT patients and 60 healthy controls were enrolled in the study. Disease severity assessment included clinical evaluation with CMT Neuropathy Score version 2 (CMTNSv2). Blood plasma NfL concentrations were measured using the single molecule array (Simoa) NfL assay. RESULTS: The NfL concentration was significantly higher in the CMT patient group than in the controls (p<0.001). Of the CMT patients, ones with type CMTX1 had a higher NfL level than those in the two other analysed subgroups (CMT1A and other CMT types) (p=0.0498). The NfL concentration had a significant but weak correlation with the CMTNSv2 (rs =0.25, p=0.012). In one CMT patient with an extremely elevated NfL level, overlap with chronic inflammatory demyelinating polyneuropathy was suspected. ROC analysis showed that an NfL concentration of 8.9 pg/mL could be used to discriminate CMT patients from controls, with an area under the curve of 0.881. CONCLUSIONS: Our study confirmed that the plasma NfL concentration is significantly higher in CMT patients than in controls. Plasma NfL concentration was found to significantly, albeit weakly, reflect the clinical severity of CMT. In the future, NfL may be used, either individually or collaboratively, as a biomarker in the clinical context of suspected CMT; however, several issues need to be addressed first

Hirnantia sagittifera

The brachiopod Hirnantia sagittifera (M’Coy) and trilobite morphs of the genus Mucronaspis from the topmost Ordovician Porkuni Stage of the central East Baltic are described and compared with those from the Hirnantian Stage of other regions. These important Hirnantian taxa occur in the Livonian Tongue of the Central Baltoscandian Facies Belt of the Baltic Basin, where the Porkuni Stage is represented by the non-graptolitic Kuldiga and Saldus formations. Hirnantia sagittifera appears in the lowermost part of the Porkuni Stage and is rather widely distributed in the basin in spite of its rare finds in each locality. Our study of trilobites of the genus Mucronaspis has enabled us to observe morphological changes in its exoskeleton in time and to identify a succession of five morphotypes (morphs). In some characteristics these morphs are similar to those of different alleged species of the genus Mucronaspis (M. olini, M. danai, M. ganabina, M. mucronata) but they cannot be definitely assigned to any of these species due to some variances. However, here for the first time a stratigraphically ordered collection is presented, which deserves attention in revising the taxonomy of highly variable Mucronaspis. The described brachiopods and trilobites occur mainly in the strata correlated with the Normalograptus extraordinarius graptolite Biozone. However, the uppermost finds of both taxa come from strata correlated with the N. persculptus Biozone

A simple lift-off-based patterning method for micro- and nanostructuring of functional substrates for cell culture

The ability to produce cell patterning through precise surface engineering has stimulated the development of cellular bioassays that offer new insights on the mechanisms of cell adhesion, proliferation, differentiation and molecular signaling pathways. Here we describe a simple micropatterning technique combining supersonic cluster beam deposition of nanostructured titania films on bovine serum albumin functionalized substrates. A standard lift-off process enables us to generate complementary micropatterns of hydrophobic bovine serum albumin (cell-repellent) and hydrophilic nanostructured TiO(x) (cell-adhesive). We demonstrate the selective PC12 cell adhesion and growth on biocompatible nanostructured TiO(x). We also observed that these functional micropatterned substrates promote a considerable enhancement of cell attachment and proliferation

Association of variants in the CP, ATOX1 and COMMD1 genes with Wilson disease symptoms in Latvia

Wilson’s disease (WD) is a copper metabolism disorder, caused by allelic variants in the ATP7B gene. Wilson’s disease can be diagnosed by clinical symptoms, increased copper and decreased cerulopasmin levels, which could all also be by other genetic variants beyond the ATP7B gene, e.g., disturbed ceruloplasmin biosynthesis can be caused by pathogenic allelic variants of the CP gene. Copper metabolism in the organism is affected by several molecules, but pathogenic variants and related phenotypes are described with COMMD1 and ATOX1 genes. The aim of the study was to test other genes, CP, ATOX1 and COMMD1, for possible influence to the manifestation of WD. Patients were enrolled on the basis of Leipzig’s diagnostic criteria, 64 unrelated patients with confirmed WD. Direct sequencing of promoter region of the CP gene and ATOX1 and COMMD1 gene exons was conducted. Statistically significant differences were found between the two variants in the CP gene and the ATP7B genotype (rs66508328 variant AA genotype and the rs11708215 variant GG genotype) were more common in WD patients with an unconfirmed ATP7B genotype. One allelic (intronic) variant was found in the ATOX1 gene without causing the functional changes of the gene. Three allelic variants were identified in the COMMD1 gene. No statistically significant differences were found between allele and genotype frequencies and the first clinical manifestations of WD. Different variants of the CP gene contributed to a WD-like phenotype in clinically confirmed WD patients with neurological symptoms and without identified pathogenic variants in the ATP7B gene. Allelic variants in the ATOX1 and COMMD1 genes do not modify the clinical manifestation of WD in Latvian patients. (266 words