Search

For some Gram-positive genera and species, the long-extended and adhesive sortase-dependent pilus plays an essential role during host colonization, biofilm formation, and immune modulation. Lactobacillus rhounnosus GG is a gut-adapted commensal strain that harbors the operonic genes for the SpaCBA and SpaFED pili, both being comprised of three different protein subunits termed the backbone, tip, and basal pilins. Crystal structures of the backbone pilins (SpaA and SpaD) have recently been solved, and here we describe the high-resolution (1.5 angstrom) structural determination of the SpaE basal pilin. SpaE consists of two immunoglobulin-like CnaB domains, with each displaying a spontaneously formed internal isopeptide bond, though apparently slow forming in the N-terminal domain. Remarkably, SpaE contains an atypically lengthy unstructured C-terminal tail, along with an YPKN pilin motif peptide, which is normally reserved for backbone subunits. Based on our analysis of the crystal structure data, we provide a molecular model for the basal positioning of the SpaE pilin within the SpaFED pilus. Non

Background: While anecdotal evidence has long claimed that a raw meat-based diet (RMBD) improves the metabolic health of canines, no rigorous scientific study has clarified this issue. Canine atopic dermatitis (CAD) has also been linked to metabolic health, but its relation to diet remains poorly understood. This study investigates whether dietary choice is linked to metabolic health in healthy and CAD-diagnosed canines via targeted serum and urine metabolomic analysis of polar, non-ionic metabolites, as well as whether the underlying CAD condition modulates the response to nutritional intake. Materials and Methods: Serum metabolites of client-owned Staffordshire bull terriers, divided into CAD-diagnosed (n = 14) and healthy (n = 6) cohorts, were studied. Urine metabolites of a subset of the CAD-diagnosed canines (n = 8) were also studied. The canines were split into two cohorts based on diet. The first cohort were fed a commercially available high-fat, moderate-protein, low-carbohydrate RMBD (n = 11, CAD diagnosed n = 8, healthy n = 3). Those in the second cohort were fed a commercially available moderate-fat, moderate-protein, high-carbohydrate kibble diet (KD) (n = 9: CAD diagnosed n = 6, healthy n = 3). The diet intervention period lasted approximately 4.5 months (median 135 days). Statistical analyses of the serum profiles across all dogs (n = 20) and the urine profiles of the CAD-diagnosed subset (n = 8) were performed. Results and Discussion: The KD cohort was found to have higher concentrations of methionine than the RMBD cohort, both in serum (all dogs, p < 0.0001) and in urine (CAD-only cohort, p < 0.0002), as well as cystathionine and 4-pyridoxic acid. Methionine plays important roles in homocysteine metabolism, and elevated levels have been implicated in various pathologies. The CAD (n = 14) cohort dogs showed starker metabolic changes in response to diet regarding these pathways compared to the healthy (n = 6) cohort. However, there was no significant change in CAD severity as a result of either diet. Likely due to the higher meat content of the RMBD, higher concentrations of several carnitines and creatine were found in the RMBD cohort. Citrulline was found in higher concentrations in the KD cohort. Our findings provide insight into the relationship between diet and the serum and urine metabolite profiles of canines. They also suggest that neither diet significantly affected CAD severity.

A gastrointestinal outbreak was reported among 154 diners who attended a Christmas buffet on the 9 and 10 December 2016. A retrospective cohort study was undertaken. Faecal samples, water, ice and an air ventilation device were tested for indicators and routine pathogens. Altogether 26% (24/91) fulfilled the case definition of having typical viral gastrointestinal symptoms. Norovirus genogroup I was detected in faecal samples from three cases. One of these cases tested positive also for sapovirus and had a family member testing positive for both norovirus and sapovirus. A diner who drank water or drinks with ice cubes (risk ratios (RR) 6.5, 95% confidence intervals (CI) 1.5–113.0) or both (RR 8.2, 95% CI 1.7–145.5) had an increased risk in a dose-response manner. Ice cubes from three vending machines had high levels of heterotrophic bacteria. A faulty air ventilation valve in the space where the ice cube machine was located was considered a likely cause of this outbreak. Leaking air ventilation valves may represent a neglected route of transmission in viral gastrointestinal outbreaks.

Yersinia pseudotuberculosis is a bacterium that not only survives, but also thrives, proliferates, and remains infective at cold-storage temperatures, making it an adept foodborne pathogen. We analyzed the differences in gene expression between Y. pseudotuberculosis IP32953 grown at 3 and 28 degrees C to investigate which genes were significantly more expressed at low temperature at different phases of growth. We isolated and sequenced the RNA from six distinct corresponding growth points at both temperatures to also outline the expression patterns of the differentially expressed genes. Genes involved in motility, chemotaxis, phosphotransferase systems (PTS), and ATP-binding cassette (ABC) transporters of different nutrients such as fructose and mannose showed higher levels of transcripts at 3 degrees C. At the beginning of growth, especially genes involved in securing nutrients, glycolysis, transcription, and translation were upregulated at 3 degrees C. To thrive as well as it does at low temperature, Y. pseudotuberculosis seems to require certain cold shock proteins, especially those encoded by yptb3585, yptb3586, yptb2414, yptb2950, and yptb1423, and transcription factors, like Rho, IF-1, and RbfA, to maintain its protein synthesis. We also found that genes encoding RNA-helicases CsdA (yptb0468), RhlE (yptb1214), and DbpA (yptb1652), which unwind frozen secondary structures of nucleic acids with cold shock proteins, were significantly more expressed at 3 degrees C, indicating that these RNA-helicases are important or even necessary during cold. Genes involved in excreting poisonous spermidine and acquiring compatible solute glycine betaine, by either uptake or biosynthesis, showed higher levels of transcripts at low temperatures. This is the first finding of a strong connection between the aforementioned genes and the cold adaptation of Y. pseudotuberculosis. Understanding the mechanisms behind the cold adaptation of Y. pseudotuberculosis is crucial for controlling its growth during cold storage of food, and will also shed light on microbial cold adaptation in general.

One promising series of small-molecule orexin receptor agonists has been described, but the molecular pharmacological properties, i.e. ability and potency to activate the different orexin receptor-regulated signal pathways have not been reported for any of these ligands. We have thus here assessed these properties for the most potent ligand of the series, 4'-methoxy-N,N-dimethyl-3'4N-(3-{[2-(3-methylbenzamido)ethyl]amino}phenyl sulfamoy1]-(1,1'-biphenyl)-3-carboxamide (Nag 26). Chinese hamster ovary-K1 cells expressing human orexin receptor subtypes OX1 and OX2 were used. Ca2+ elevation and cell viability and death were assessed by fluorescent methods, the extracellular signal-regulated kinase pathway by a luminescent Elk-1 reporter assay, and phospholipase C and adenylyl cyclase activities by radioactive methods. The data suggest that for the G(q)-dependent responses, Ca2+, phospholipase C and Elk-1, Nag 26 is a full agonist for both receptors, though of much lower potency. However, saturation was not always reached for OX1, partially due to Nag 26s low solubility and partially because the response decreased at high concentrations. The latter occurs in the same range as some reduction of cell viability, which is independent of orexin receptors. Based on the EC50, Nag 26 was OX2 selective by 20-200 fold in different assays, with some indication of biased agonism (as compared to orexin-A). Nag 26 is a potent orexin receptor agonist with a largely similar pharmacological profile as orexin-A. However, its weaker potency (low-mid micromolar) and low water solubility as well as the non-specific effect in the mid-micromolar range may limit its usefulness under physiological conditions.

Introduction: Influenza-associated severe acute respiratory infections (SARI) are a major contributor to global morbidity and mortality. In response to a cluster of SARI cases and deaths in pregnant women, with two deceased cases testing positive for influenza A(H1N1) pdm09, an investigation was initiated to determine whether there was an increase of paediatric SARI cases admitted to divisional hospital intensive care units in Fiji in may 2016 compared to May 2013-2015. Methods: Retrospective case finding was conducted at the paediatric intensive care units (PICUs) in Fiji's three divisional hospitals. Data were collected from 1 January 2013 to 26 May 2016. Cases were identified using a list of clinical diagnoses compatible with SARI. Results: A total of 632 cases of paediatric SARI with complete details were identified. The median age of cases was 6 months (Interquartile range: 2-14 months). Children aged less than 5 years had a higher rate of paediatric SARI requiring admission to a divisional hospital PICU in May 2016 compared to May 2013-2015 (Incidence rate ratio: 1.7 [95% CI: 1.1-2.6]). This increase was not observed in children aged 5-14 years. The case-fatality ratio was not significantly different in 2016 compared to previous years. Conclusion: The investigation enabled targeted public health response measures, including enhanced SARI surveillance at divisional hospitals and an emergency influenza vaccination campaign in the Northern Division.

Background Several eastern European countries reported a severe influenza season to the World Health Organization (WHO) during late 2015. A country-specific rapid risk assessment for Ukraine was conducted to assess the season's severity and inform public health action. Methods The exposure and hazard were assessed using acute respiratory infection (ARI), severe acute respiratory infection (SARI), laboratory surveillance, virological and vaccine data from weeks 40/2015 to 7/2016 with comparison to 4 previous seasons to describe the influenza season start (5-week consecutive increase in ARI or SARI), predominant virus types, geographical spread and affected age groups. Results The exposure was characterised by an earlier and steeper increase in SARI (week 1/2016) and ARI (week 2/2016) compared to the previous 4 seasons. Transmission was across Ukraine with an increase in ARI and SARI cases aged 30-64 years compared to 2014/15. Laboratory-confirmed deaths increased from 11 in 2014/2015 to 342 in 2015/2016; the majority were 30-64 years old and unvaccinated; and 63.5% had underlying conditions. Total population vaccination coverage was 0.3%. The hazard assessment found influenza virus A(H1N1)pdm09 accounted for >95% of viruses detected. Ukrainian virus strains (n = 62) were antigenically similar to vaccine strains and susceptible to neuraminidase inhibitors. Conclusions The first weeks of the 2015/16 influenza season were more severe than previous seasons, with an earlier and steeper increase in severe cases and deaths, particularly in younger adults. Influenza A(H1N1)pdm09 was the predominant strain and was closely related to the seasonal vaccine strain with no evidence of resistance to antiviral drugs.

Bacterial genome sequencing has led to the development of new approaches for the analysis of food-borne epidemics and the exploration of the relatedness of outbreak-associated isolates and their separation from nonassociated isolates. Using Illumina technology, we sequenced a total of six isolates (two from patients, two from raw bulk milk, and two from dairy cattle) associated with a milk-borne Campylobacter jejuni outbreak in a farming family and compared their genomes. These isolates had identical pulsed-field gel electrophoresis (PFGE) types, and their multilocus sequence typing (MLST) type was ST-50. We used the Ma_1 isolate (milk) as the reference, and its genome was assembled and tentatively ordered using the C. jejuni NCTC 11168 genome as the scaffold. Using whole-genome MLST (wgMLST), we identified a total of three single-nucleotide polymorphisms (SNPs) and differences in poly(G or C) or poly(A or T) tracts in 12 loci among the isolates. Several new alleles not present in the database were detected. In contrast, the sequences of the unassociated C. jejuni strains P14 and 1–12S (both ST-50) differed by 420 to 454 alleles from the epidemic-associated isolates. We found that the fecal contamination of bulk tank milk occurred by highly related sequence variants of C. jejuni , which are reflected as SNPs and differences in the length of the poly(A or T) tracts. Poly(G or C) tracts are reversibly variable and are thus unstable markers for comparison. Further, unrelated strains of ST-50 were clearly separated from the outbreak-associated isolates, indicating that wgMLST is an excellent tool for analysis. In addition, other useful data related to the genes and genetic systems of the isolates were obtained.

Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide. Despite advances in combined modality therapy (surgery, radiotherapy, chemotherapy) the 5-year survival rate in stage III and IV disease remains at 40% - 60%. Short-range Auger-electron emitters, such as In-111 and In-114m, tagged with a drug, molecule, peptide, protein or nanoparticles brought in close proximity to nuclear DNA represent a fascinating alternative for treating cancer. In this thesis, we studied the usefulness of Indium-111-bleomycin complex (In-111-BLMC) in the diagnostics and potential therapy of HNSCC using in vitro HNSCC cell lines, in vivo nude mice, and in vivo HNSCC patients. In in vitro experiments with HNSCC cell lines, the sensitivity to external beam radiation, BLM, In-111-BLMC, and In-111-Cl3 was studied using the 96-well plate clonogenic assay. The influence of BLM and In-111-BLMC on the cell cycle was measured with flow cytometry. In in vivo nude mice xenograft studies, the activity ratios of In-111-BLMC were obtained in gamma camera images. The effect of In-111-BLMC in HNSCC xenografts was studied. In in vivo patient studies, we determined the tumor uptake of In-111-BLMC with gamma camera and the radioactivity from tumor samples using In-111-BLMC with specific activity of 75, 175, or 375 MBq/mg BLM. The S values, i.e. absorbed dose in a target organ per cumulated activity in a source organ, were simulated for In-111 and In-114m. In vitro studies showed the variation of sensitivity for external beam radiation, BLM, and In-111-BLMC between HNSCC cell lines. IC50 values for BLM were 1.6-, 1.8-, and 2.1-fold higher than In-111-BLMC (40 MBq/mg BLM) in three HNSCC cell lines. Specific In-111 activity of 40 MBq/mgBLM was more effective in killing cells than specific In-111 activity of 195MBq/mgBLM (p=0.0023). In-111-Cl3 alone had no killing effect. The percentage of cells in the G2/M phase increased after exposure to BLM and especially to In-111-BLMC in the three cell lines studied, indicating a G2/M block. The tumor-seeking behavior was shown in the in vivo imaging study of xenografted mice. BLM and In-111-BLMC were more effective than NaCl in reducing xenografted tumor size in HNSCC. The uptake ratios received from gamma images in the in vivo patient study varied from 1.2 to 2.8 in malignant tumors. However, the uptake of In-111-BLMC was unaffected by increasing the injected activity. A positive correlation existed between In-111-BLMC uptake, Ki-67/MIB activity, and number of mitoses. Regarding the S values, In-114m delivered a 4-fold absorbed radiation dose into the tumor compared with In-111, and thus, In-114m-BLMC might be more effective than In-111-BLMC at the DNA level. Auger-electron emitters, such as In-111 and In-114m, might have potential in the treatment of HNSCC. Further studies are needed to develop a radiopharmaceutical agent with appropriate physical properties of the radionuclide and a suitable carrier to bring it to the targeted tissue. Nanoalueen kantaman Auger-elektronisäteilijä syövän hoitoon Auger-elektroneja säteilevä radioaktiivinen isotooppi, liitettynä syöpäsoluun hakeutuvaan sytostaattiin, vasta-aineeseen tai esim. nanopartikkeliin, on potentiaalinen tulevaisuuden syövän hoitomuoto. Auger-elektronien kantama on lyhyt, suurimmillaan n. 0,5 millimetrin miljoonasosaa. Syöpäsoluun tunkeutunut Auger-elektronisäteilijä tuhoaa solun DNA:ta mainitun kantaman sisällä. Pään ja kaulan alueen syövistä noin 90% on levyepiteelisolusyöpiä. Potilaan, jolla on paikallisesti tai laajemmalle levinnyt levyepiteelisyöpä, 5-vuotis eloonjäämisennuste jää nykyisillä yhdistelmähoidoilla (kirurgia, sädehoito, kemoterapia) 40% - 60%:iin. Väitöskirjatutkimuksessa tutkin soluviljely-, eläin- ja potilastöin Indium-111:n (In-111) ja bleomysiinin:n (BLM) yhdistelmän, indium-111-bleomysiinikompleksin (In-111-BLMC), soveltuvuutta levyepiteelisyövän diagnostiikkaan ja hoitoon. BLM on solua tuhoava sytostaatti. Radioaktiivinen isotooppi In-111, jonka puoliintumisaika on 2,8 vuorokautta, säteilee gamma- ja röntgensäteiden lisäksi beta-partikkeleita ja Auger-elektroneja. Soluviljelytöissä In-111-BLMC tuhosi potilaiden syöpäkudoksista eristettyjä syöpäsoluja merkittävästi tehokkaammin kuin pelkkä BLM. Yllättävä tulos oli, että radioaktiivisuudeltaan miedompi In-111-BLMC (40 MBq/mg BLM) oli tehokkaampi syöpäsolujen tuhoaja kuin aktiivisuudeltaan voimakkaampi In-111-BLMC (195 MBq/mg BLM). Mielenkiintoinen tulos oli, että BLM ja varsinkin In-111-BLMC aiheuttivat häiriöitä syöpäsolujen solunjakautumiseen. Koe-eläintutkimuksissa todettiin In-111-BLMC:n hakeutuminen syöpäkudokseen. Hoitokokeessa ihonalaiset syövät pienenivät BLM:a sekä In-111-BLMC:a saaneilla hiirillä. Kymmenelle potilaalle annettiin suoneen vaihtoehtoisesti kolme eri radioaktiivisuuden omaavaa diagnostista annosta In-111-BLMC:a. Gammakamerakuvaus osoitti In-111-BLMC:n hakeutuvan syöpäkudokseen. Syövät kuvautuivat gammakamerakuvissa pienemmälläkin tutkitulla aktiivisuusannoksella. Leikkauksessa otettujen syöpäkudospalojen mitatut radioaktiivisuudet korreloivat gammakamerakuvista laskettuihin kertymiin sekä solunjakautumista kuvaavaan mitoosien määrään. Syöpäpotilaan CT-kuvien perusteella mallinnettiin potilaan suun ja kaulan alueen geometria. Monte Carlo-menetelmällä simuloitiin radioaktiivisen säteilyn fotonien liike sekä laskettiin elimistön eri kudosten säteilyannoksia kuvaavat S-arvot In-111:lle ja In-114m:lle. In-114m on indiumin yksi isotooppi, joka on myös Auger-elektronisäteilijä ja sen puoliintumisaika on 49,5 vuorokautta. In-114m luovutti 4-kertaa suuremman säteilyannoksen kasvaimeen kuin In-111 ja voisi olla In-111:a tehokkaampi isotooppi. Syöpäsolun DNA:n läheisyyteen viety Auger-elektronisäteilijä, esim. In-111 ja In-114m, on potentiaalinen vaihtoehto syövän hoidossa tulevaisuudessa, jonka ominaisuudet saattaisivat myös vähentää hoidon haittavaikutuksia.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time. Kasvohermohalvauksen kirurginen hoito hermotetulla lihassiirteellä Pitkäaikainen kasvohermohalvaus on potilaalle henkisesti raskas ja kuormittava sairaus, sillä se vaikeuttaa merkittävästi sosiaalista kanssakäymistä ja aiheuttaa suuren kosmeettisen ja toiminnallisen haitan näille potilaille. Osa näistä potilaista hyötyy leikkaushoidosta, ja lisätiedon saanti leikkaustulokseen vaikuttavista tekijöistä on ensiarvoisen tärkeää, jotta oikeat potilaat voidaan valita ja leikkausmenetelmiä parantaa. Pitkäaikaisen kasvohermohalvauksen paras tunnettu hoitomuoto on hermotettu vapaa lihassiirre. Se mahdollistaa kasvojen lihasten toiminnan ja tunteiden ilmaisun.Tässä kaksivaiheisessa leikkauksessa tuodaan ensin vastakkaiselta puolelta kasvoja terveestä kasvohermosta hermosiirrettä käyttäen hermotus halvaantuneelle puolelle. Toisessa leikkauksessa noin puoli vuotta myöhemmin siirretään muualta kehosta vapaa lihassiirre. Tämän lihassiirteen oma hermo yhdistetään aiemmin halvaantuneelle puolelle tuotuun hermosiirteeseen ja verisuonet yhdistetään paikalla oleviin verisuoniin. Optimaalisessa tapauksessa tämä hermotettu lihassiirre alkaa toimia korvaten halvaantuneet kasvojen lihakset, ja potilas saa ilmeensä ja hymynsä takaisin. Tässä väitöskirjatyössä selvitettiin tämän leikkaushoidon pitkäaikaistuloksia ja potilastyytyväisyyttä sekä niitä tekijöitä, jotka vaikuttavat toiminnalliseen lopputulokseen. Potilaista noin 60% saavutti hyvän kasvojen lihassymmetrian ja toiminnallisen tuloksen, ja 78% koki elämänlaatunsa parantuneen selvästi. Pitkä seuranta-aika leikkauksen jälkeen heikensi toiminnallista tulosta. Magneettikuvauksen perusteella lihassiirre kutistui merkittävästi. Magneettikuvauksessa normaalilta näyttävä lihassiirre myös toimi hyvin. Lihas- ja hermossiirteen koepaloista selvitimme, että sekä lihassiirteen säikeet että hermosiirteen toimivien hermojen määrä oli merkittävästi vähentynyt myös silloin, kun lihassiirteen toiminta oli hyvä. Kaikista hermossiirteistä löysimme hermokasvutekijäreseptoria (p75NGFR) ja verisuonikasvutekijää (VEGF), mutta terveistä hermoista näitä ei löytynyt.Hermokasvutekijäreseptorin ilmeneminen oli yhteydessä hyvään toiminnalliseen tulokseen. Aiemmin näiden kasvutekijöiden ilmenemistä ei ole kuvattu ihmisen hermosiirteissä. Tässä väitöskirjatyössä etsimme selittäviä tekijöitä hyvälle tai heikolle lihastoiminnalle vapaassa, hermotetussa lihassiirteessä. Lisäksi tutkimustuloksemme valottavat yleisimminkin vapaan hermosiirteen sekä vapaan hermotetun lihassiirteen piirteitä ja kasvutekijöiden ilmenemistä niissä. Tulevaisuudessa kasvutekijähoidot saattavat auttaa hermoston sairauksista tai vammoista kärsiviä potilaita.