UriSed 3 PRO automated microscope in screening bacteriuria at region-wide laboratory organization

Background and aims: We assessed the possibility to rule out negative urine cultures by counting with UriSed 3 PRO (77 Elektmnika, Hungary) at Helsinki and Uusimaa Hospital District. Materials and methods: Bacteria counting of the UriSed 3 PRO automated microscope was verified with reference phase contrast microscopy against growth in culture. After acceptance into routine, results of bacteria and leukocyte counting from 56 426 specimens with eight UriSed 3 PRO instruments were compared against results from parallel samples cultured on chromogenic agar. Laboratory data including preanalytical details were accessed through the regional database of the Helsinki and Uusimaa Hospital District. Results: A combined sensitivity of 87-92% and a negative predictive value of 90-96% with a specificity of 54-50% was reached, depending on criteria. Preanalytical data (incubation time in bladder) combined with the way of urine collection would improve these figures if reliable. Conclusions: Complex patient populations, regional logistics and data interfases, and economics related to increased costs of additional particle counts against costs of screening cultures of all samples, did not support adaptation of a screening process of urine cultures. This conclusion was made locally, and may not be valid elsewhere.Peer reviewe

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH

Peer reviewe

Hijacking the human complement inhibitor C4b-binding protein by the sporozoite stage of the Plasmodium falciparum parasite

The complement system is considered the first line of defense against pathogens. Hijacking complement regulators from blood is a common evasion tactic of pathogens to inhibit complement activation on their surfaces. Here, we report hijacking of the complement C4b-binding protein (C4bp), the regulator of the classical and lectin pathways of complement activation, by the sporozoite (SPZ) stage of the Plasmodium falciparum parasite. This was shown by direct binding of radiolabeled purified C4bp to live SPZs as well as by binding of C4bp from human serum to SPZs in indirect immunofluorescence assays. Using a membrane-bound peptide array, peptides from the N-terminal domain (NTD) of P. falciparum circumsporozoite protein (CSP) were found to bind C4bp. Soluble biotinylated peptide covering the same region on the NTD and a recombinantly expressed NTD also bound C4bp in a dose-dependent manner. NTD-binding site on C4bp was mapped to the CCP1-2 of the C4bp alpha-chain, a common binding site for many pathogens. Native CSP was also co-immunoprecipitated with C4bp from human serum. Preventing C4bp binding to the SPZ surface negatively affected the SPZs gliding motility in the presence of functional complement and malaria hyperimmune IgG confirming the protective role of C4bp in controlling complement activation through the classical pathway on the SPZ surface. Incorporating the CSP-C4bp binding region into a CSP-based vaccine formulation could induce vaccine-mediated immunity that neutralizes this immune evasion region and increases the vaccine efficacy.Peer reviewe

Comprehensive development of nearly zero-energy municipal service buildings (COMBI). Tutkimushankkeen johdanto- ja yhteenvetoraportti.

Tässä COMBI-tutkimushankkeen johdanto- ja yhteenvetoraportissa esitetään vuosina 2015—2018 toteutetun tutkimushankkeen keskeiset suositukset ja johtopäätökset. Hankkeen tavoitteena on ollut parantaa julkisten palvelurakennusten, kuten koulujen, päiväkotien ja vanhainkotien energiatehokkuutta turvallisesti ja kustannustehokkaasti. Hankkeessa on tarkasteltu sekä uudis- että korjausrakentamista.Tutkittuja aihepiirejä on hankkeessa ollut suuri määrä. Arkkitehtuurin osalta on tarkasteltu palvelurakennusten arkkitehtisuunnittelun kehittämistä energiatehokkuuden ja tilasuunnittelun näkökulmista sekä ympäristöystävällisyyden ja kestävyyden huomioon ottamista arkkitehtisuunnittelussa. Rakenteiden osalta on tutkittu niiden lämpö- ja kosteusteknistä toimintaa ja määritetty kosteusteknisiä materiaaliominaisuuksia. Kenttätutkimuksissa on tarkasteltu sisäilman olosuhteita 24 palvelurakennuksessa Tampereen ja ympäristökuntien sekä Helsingin alueella. Myös palvelurakennusten laskennallista ja toteutunutta energiankulutusta on tutkittu Tampereen, Helsingin ja Oulun kohteista. Taloteknisten järjestelmien osalta on tarkasteltu niiden kustannusoptimaalisuutta, uusiutuvan energian etätuotantoa, taloautomaatiojärjestelmien toimintaa, aurinkosuojausta ja valaistusta. Rakennusprosessin osalta näkökulmina ovat olleet päätöksenteon prosessit, talotekniikan käytännön toteutus, rakennuksen toimivuuden varmistus sekä olosuhteiden ja energiankulutuksen seuranta. Lisäksi on kehitetty työkaluja rakennushankkeen taloudellisuustarkasteluihin.Tämän johdanto- ja yhteenvetoraportin liitteenä on hankkeen tulosten pohjalta koottu COMBI 8 suosituslista, jossa esitettyjen toimenpiteiden katsotaan laajasti edesauttavan julkisten palvelurakennusten toimivuutta ja energiatehokkuutta. Raportin liitteenä on myös 45 kpl lyhyitä tuloskortteja sekä niihin liittyvät esitysaineistot, joiden tarkoituksena on helpottaa hankkeessa kerätyn tiedon leviämistä. Hankkeen alkuperäisjulkaisut on listattu tämän raportin liitteenä olevassa julkaisuluettelossa.Keskeisenä johtopäätöksenä todetaan, että hyvä energiatehokkuus on ainoastaan yksi laadukkaan rakentamisen monista ominaisuuksista. Laadukas rakentaminen edellyttää kokonaisvaltaista ja oikeaaikaista asioiden tarkastelua sekä ehjän ketjun rakentamista suunnittelusta toteutukseen ja käyttöön. Tässä onnistumisen edellytyksenä ovat rakennushankkeessa ja sen jälkeen rakennuksen parissa toimivien henkilöiden hyvä ammattitaito ja yhteistyö sekä riittävät resurssit. COMBI-hankkeen tulosten tavoitteena on antaa eri osapuolille tietoa ja työkaluja turvallisen, taloudellisen ja energiatehokkaan lopputuloksen saavuttamiseksi

New tools for the study of an old collagen:characterization of the human COL9A1, COL9A2 and COL9A3 genes and production of human type IX collagen as a recombinant protein

Abstract Type IX collagen is a quantitatively minor component of cartilage collagen fibrils. Although a few mutations have been associated with multiple epiphyseal dysplasia, recent evidence suggests involvement of type IX collagen in a wider spectrum of phenotypes. The functional role of this molecule remains undetermined, in part due to difficulties in obtaining high amounts of intact protein. To facilitate more efficient mutation screening and comparison of the genomic organization of the human genes encoding the α1(IX), α2(IX) and α3(IX) polypeptides, their genomic structures were characterized. Complete nucleotide sequences were determined for the COL9A2 and COL9A3 genes along with sequences for all the exon boundaries in the COL9A1 gene. Putative transcription control elements were identified and the alternative promoter region was characterized in the human and mouse COL9A1 genes. Mutation screening was performed for the COL9A3 gene and two apparently neutral 9-bp deletions within the COL1 domain were identified. These are the first deletions within a triple-helical domain of any collagen that are not associated with a disease phenotype. An insect cell expression system with an exogenous source of prolyl 4-hydroxylase was used to produce heterotrimeric human type IX collagen. The recombinant protein consisted of the three a chains in a 1:1:1 ratio and showed correct folding and high thermal stability. Up to 10 mg of secreted protein could be purified from a litre of culture medium. The expression system was used to analyze the chain association of type IX collagen in cellulo. Although the chains are capable of homotrimerization, a preference for heterotrimer formation was noted.The neutral deletion was characterized further using the insect cell system. Mutant α3(IX) chains carrying a deletion of one Gly-X-Y triplet were shown to form correctly folded heterotrimers with the wild-type α1(IX) and α2(IX) chains. The results suggest a function for the NC2 domain in neutralizing the effect of the deletion. This work provides a novel means for the analysis of type IX collagen mutations and their protein-level effects, and should enable future studies to be made of the structure-function relationship in type IX collagen

Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from <i>S. erythraea</i>:A molten globule when deprived of Ca²⁺

Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca2+ and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca2+-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca2+-saturated states

Verification of UriSed 3 PRO automated urine microscope in regional laboratory environment

Background and aims: Ten UriSed 3 PRO automated microscopes (77 Elektronika, Hungary) were verified for nine HUSLAB laboratories with 160 000 annual urine samples. Materials and methods: Particle counting of the primary UriSed 3 PRO instrument (77 Elektronika, Hungary) was verified against reference visual microscopy with 463 urine specimens, and against urine culture on chromogenic agar plates with parallel 396 specimens. Nine secondary instruments were compared pairwise with the primary instrument. Results: Relative imprecisions compared to Poisson distribution, R(CV), were estimated to be 1.0 for white blood cell (WBC) and 1.5 for red blood cell (RBC) counts, respectively. Spearman's correlations against visual microscopy were rS = 0.94 for WBC, rS = 0.87 for RBC, and rS = 0.82 for squamous epithelial cell (SEC) counts. Agreement with visual microscopy (Cohen's weighted kappa) was 0.94 for WBC, 0.89 for RBC, 0.88 for SEC, 0.59 for combined casts, and 0.49 for non-squamous epithelial cells (NEC). Bacteria were detected with a sensitivity of 90% and specificity of 39 against culture at 107 CFB/L (104 CFU/mL). Created flagging limits allowed automated reporting for 70-75% of patient results. Conclusions: UriSed 3 PRO instruments were adopted into routine use after acceptance of the verification.Peer reviewe

Mutations in cartilage oligomeric matrix protein causing pseudoachondroplasia and multiple epiphyseal dysplasia affect binding of calcium and collagen I, II, and IX

Mutations in type 3 repeats of cartilage oligomeric matrix protein (COMP) cause two skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We expressed recombinant wild-type COMP that showed structural and functional properties identical to COMP isolated from cartilage. A fragment encompassing the eight type 3 repeats binds 14 calcium ions with moderate affinity and high cooperativity and presumably forms one large disulfide-bonded folding unit. A recombinant PSACH mutant COMP in which Asp-469 was deleted (D469 Delta) and a MED mutant COMP in which Asp-361 was substituted by Tyr (D361Y) were both secreted into the cell culture medium of human cells. Circular dichroism spectroscopy revealed only small changes in the secondary structures of D469 Delta and D361Y, demonstrating that the mutations do not dramatically affect the folding and stability of COMP. However, the local conformations of the type 3 repeats were disturbed, and the number of bound calcium ions was reduced to 10 and 8, respectively. In addition to collagen I and II, collagen IX also binds to COMP with high affinity. The PSACH and MED mutations reduce the binding to collagens I, II, and IX and result in an altered zinc dependence. These interactions may contribute to the development of the patient phenotypes and may explain why MED can also be caused by mutations in collagen IX genes