Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.

BACKGROUND: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrPd). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC. METHODS: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP. RESULTS: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution. CONCLUSIONS: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Potentially Inappropriate Prescribing and Potential Prescribing Omissions in 82,935 Older Hospitalised Adults: Association with Hospital Readmission and Mortality Within Six Months.

Polypharmacy with "potentially inappropriate medications" (PIMs) and "potential prescribing omissions" (PPOs) are frequent among those 65 and older. We assessed PIMs and PPOs in a retrospective study of 82,935 patients ≥ 65 during their first admission in the period March 2013 through February 2018 to the four acute-care Calgary hospitals. We used the American Geriatric Society (AGS) and STOPP/START criteria to assess PIMs and PPOs. We computed odds ratios (ORs) for key outcomes of concern to patients, their families, and physicians, namely readmission and/or mortality within six months of discharge, and controlled for age, sex, numbers of medications, PIMs, and PPOs. For readmission, the adjusted OR for number of medications was 1.09 (1.09-1.09), for AGS PIMs 1.14 (1.13-1.14), for STOPP PIMs 1.15 (1.14-1.15), for START PPOs 1.04 (1.02-1.06), and for START PPOs correctly prescribed 1.16 (1.14-1.17). For mortality within 6 months of discharge, the adjusted OR for the number of medications was 1.02 (1.01-1.02), for STOPP PIMs 1.07 (1.06-1.08), for AGS PIMs 1.11 (1.10-1.12), for START PPOs 1.31 (1.27-1.34), and for START PPOs correctly prescribed 0.97 (0.94-0.99). Algorithm rule mining identified an 8.772 higher likelihood of mortality with the combination of STOPP medications of duplicate drugs from the same class, neuroleptics, and strong opioids compared to a random relationship, and a 2.358 higher likelihood of readmission for this same set of medications. Detailed discussions between patients, physicians, and pharmacists are needed to improve these outcomes.

Pelvic floor disorders in women who consult primary care clinics: development and validation of case definitions using primary care electronic medical records.

BACKGROUND: To date, there has been no validated method to identify cases of pelvic floor disorders in primary care electronic medical record (EMR) data. We aimed to develop and validate symptom-based case definitions for urinary incontinence, fecal incontinence and pelvic organ prolapse in women, for use in primary care epidemiologic or clinical research. METHODS: Our retrospective study used EMR data from the Southern Alberta Primary Care Research Network (SAPCReN) and the Canadian Primary Care Sentinel Surveillance Network (CPCSSN) in southern Alberta. Trained researchers remotely reviewed a random sample of EMR charts of women aged 18 years or older from 6 rural and urban clinics to validate case definitions for urinary incontinence, fecal incontinence and pelvic organ prolapse. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and estimated SAPCReN prevalence as appropriate. RESULTS: Charts of 900 women were included. Sensitivity was 81.9% (95% confidence interval [CI] 75.1-87.2) for urinary incontinence, 61.2% (95% CI 46.2-74.5) for fecal incontinence, and 51.8% (95% CI 40.6-62.8) for pelvic organ prolapse. Corresponding specificity values were 71.9% (95% CI 68.4-75.1), 99.2% (95% CI 98.2-99.6) and 98.8% (95% CI 97.7-99.4), PPVs 40.6% (95% CI 35.4-46.0), 81.1% (95% CI 64.3-91.4) and 81.1% (95% CI 67.6-90.1), and NPVs 94.4% (95% CI 92.1-96.1), 97.8% (95% CI 96.5-98.6) and 95.3% (95% CI 93.6-96.6). The SAPCReN-observed prevalence for urinary incontinence was 29.7% (95% CI 29.3-30.0), but the adjusted prevalence was 2.97%. INTERPRETATION: The case definition for urinary incontinence met our standard for validity (sensitivity and specificity > 70%), and the case definitions for fecal incontinence and pelvic organ prolapse had PPVs greater than 80%. The urinary incontinence definition may be used in epidemiologic research, and those for fecal incontinence and pelvic organ prolapse may be used in quality-improvement studies or creation of disease registries. Our symptom-based case definitions could also be adapted for research in other EMR settings.

Advances in maize genomics: the emergence of positional cloning.

Positional cloning has been and remains a powerful method for gene identification in Arabidopsis. With the completion of the rice genome sequence, positional cloning in rice also took off, including the cloning of several quantitative trait loci. Positional cloning in cereals such as maize whose genomes are much larger than that of rice was considered near impossible because of the vast amounts of repetitive DNA. However, conservation of synteny across the cereal genomes, in combination with new maize resources, has now made positional cloning in maize feasible. In fact, a chromosomal walk is usually much faster than the more traditional method of gene isolation in maize by transposon tagging.

Implementation of data management and effect on chronic disease coding in a primary care organisation: A parallel cohort observational study.

BACKGROUND: Consistent and standardized coding for chronic conditions is associated with better care; however, coding may currently be limited in electronic medical records (EMRs) used in Canadian primary care.Objectives To implement data management activities in a community-based primary care organisation and to evaluate the effects on coding for chronic conditions. METHODS: Fifty-nine family physicians in Toronto, Ontario, belonging to a single primary care organisation, participated in the study. The organisation implemented a central analytical data repository containing their EMR data extracted, cleaned, standardized and returned by the Canadian Primary Care Sentinel Surveillance Network (CPCSSN), a large validated primary care EMR-based database. They used reporting software provided by CPCSSN to identify selected chronic conditions and standardized codes were then added back to the EMR. We studied four chronic conditions (diabetes, hypertension, chronic obstructive pulmonary disease and dementia). We compared changes in coding over six months for physicians in the organisation with changes for 315 primary care physicians participating in CPCSSN across Canada. RESULTS: Chronic disease coding within the organisation increased significantly more than in other primary care sites. The adjusted difference in the increase of coding was 7.7% (95% confidence interval 7.1%-8.2%, p < 0.01). The use of standard codes, consisting of the most common diagnostic codes for each condition in the CPCSSN database, increased by 8.9% more (95% CI 8.3%-9.5%, p < 0.01). CONCLUSIONS: Data management activities were associated with an increase in standardized coding for chronic conditions. Exploring requirements to scale and spread this approach in Canadian primary care organisations may be worthwhile.

Fluorescent protein marker lines in maize: generation and applications.

Fluorescent proteins (FP) have significantly impacted the way that we study plants in the past two decades. In the post-genomics era, these FP tools are in higher demand by plant scientists for studying the dynamics of protein localization, function, and interactions, and to translate sequence information to biological knowledge that can benefit humans. Although FP tools have been widely used in the model plant Arabidopsis, few FP resources have been developed for maize, one of the most important food crops worldwide, and an ideal species for genetic and developmental biology research. In an effort to provide the maize and cereals research communities with a comprehensive set of FP resources for different purposes of study, we generated more than 100 stable transformed maize FP marker lines, which mark most compartments in maize cells with different FPs. Additionally, we are generating driver and reporter lines, based on the principle of the pOp-LhG4 transactivation system, allowing specific expression or mis-expression of any gene of interest to precisely study protein functions. These marker lines can be used not only for static protein localization studies, but will be useful for studying protein dynamics and interactions using kinetic microscopy methods, such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and fluorescence resonance energy transfer (FRET).

The chaperonin CCT8 facilitates spread of tobamovirus infection.

The homeodomain transcription factor KNOTTED1 (KN1) functions in shoot meristem maintenance and is thought to move from cell to cell in a similar fashion as viral movement proteins. Both types of transported proteins bind to RNA, and associate with intercellular bridges formed by plasmodesmata. In a mutant screen for KN1 transport deficiency, a component of a type II chaperonin complex, CCT8, was identified, and found to interact with non-cell-autonomous proteins. The cct8 mutants are characterized by limited functionality of non-cell-autonomous proteins after their movement, and a phenotype resembling lack of homeodomain protein activity. Evidence suggests that CCT8 functions in post-translocational refolding of transported proteins. Here we show that spread of tobamovirus infection is reduced in a cct8 mutant. This suggests that similar to KN1, viral movement proteins are unfolded and refolded during transport to gain functionality in the receiving cells.

The safety of yellow fever vaccine 17D or 17DD in children, pregnant women, HIV+ individuals, and older persons: systematic review.

Yellow fever vaccine provides long-lasting immunity. Rare serious adverse events after vaccination include neurologic or viscerotropic syndromes or anaphylaxis. We conducted a systematic review of adverse events associated with yellow fever vaccination in vulnerable populations. Nine electronic bibliographic databases and reference lists of included articles were searched. Electronic databases identified 2,415 abstracts for review, and 32 abstracts were included in this review. We identified nine studies of adverse events in infants and children, eight studies of adverse events in pregnant women, nine studies of adverse events in human immunodeficiency virus-positive patients, five studies of adverse events in persons 60 years and older, and one study of adverse events in individuals taking immunosuppressive medications. Two case studies of maternal-neonate transmission resulted in serious adverse events, and the five passive surveillance databases identified very small numbers of cases of yellow fever vaccine-associated viscerotropic disease, yellow fever vaccine-associated neurotropic disease, and anaphylaxis in persons ≥ 60 years. No other serious adverse events were identified in the other studies of vulnerable groups.

An ex vivo model for suture-less amniotic membrane transplantation with a chemically defined bioadhesive.

PURPOSE: Chemically defined bioadhesives (CDB) are "two-component reactive adhesives" made from antibiotic food additives. They contain no animal-derived ingredients and are characterized by a high level of self-degradability, low toxicity, and strong bonding properties. Amniotic membrane (AM) transplantation is used to repair severe ocular surface disease in humans and horses. AM requires meticulous suturing to the injured cornea. The purpose of this study is to determine whether CDB can facilitate the suture-less placement and adherence of equine AM to porcine cadaver cornea. DESIGN: Prospective study. MATERIALS AND METHODS: To determine whether there are differences in ex vivo adhesion force between equine AM bonded with CDB to porcine cadaver cornea and equine AM sutured to porcine cadaver cornea. The following groups were tested (n = 5 per group): (1) CDB group: trephined (8 mm in diameter) cornea was bonded with CDB to 8-mm-sized discs of AM. (2) Suture group: the 8-mm trephined cornea was sutured with 8-0 absorbable suture to the AM. (3) Control group 1: two sheets of nitrocellulose paper were bonded with CDB. (4) Control group 2: two sheets of nitrocellulose paper were attached with suture and tested. Each sample was anchored to a tensiometer materials testing machine, and the strength of adhesion measured and expressed in units of kilogram-force (kgf). RESULTS: Mean ± SD strength of adhesion force found no statistically significant difference between CDB group (0.091 ± 0.054 kgf; 15 min of CDB adhesion time) and suture groups (0.095 ± 0.037 kgf), whereas there was significant difference (P < 0.05) between control group 1 (0.679 ± 0.048 kgf) and control group 2 (0.585 ± 0.092 kgf). These results revealed that the 15 min of CDB adhesive time had adhesive properties as strong as suture group. CONCLUSIONS AND CLINICAL RELEVANCE: CDB provides a satisfactory level of adherence to ex vivo suture-less attachment of equine amniotic membrane transplantation to the porcine cornea.

Reporting rates of yellow fever vaccine 17D or 17DD-associated serious adverse events in pharmacovigilance data bases: systematic review.

PURPOSE: To assess the reporting rates of serious adverse events attributable to yellow fever vaccination with 17D and 17DD strains as reported in pharmacovigilance databases, and assess reasons for differences in reporting rates. METHODS: We searched 9 electronic databases for peer reviewed and grey literature (government reports, conferences), in all languages. Reference lists of key studies were also reviewed to identify additional studies. RESULTS: We identified 2,415 abstracts, of which 472 were selected for full text review. We identified 15 pharmacovigilance databases which reported adverse events attributed to yellow fever vaccination, of which 10 contributed data to this review with about 107,600,000 patients (allowing for overlapping time periods for the studies of the US VAERS database), and the data are very heavily weighted (94%) by the Brazilian database. The estimates of serious adverse events form three groups. The estimates for Australia were low at 0/210,656 for "severe neurological disease" and 1/210,656 for YEL-AVD, and also low for Brazil with 9 hypersensitivity events, 0.23 anaphylactic shock events, 0.84 neurologic syndrome events and 0.19 viscerotropic events cases/million doses. The five analyses of partly overlapping periods for the US VAERS database provide an estimate of 3.6/cases per million YEL-AND in one analysis and 7.8 in another, and 3.1 YEL-AVD in one analysis and 3.9 in another. The estimates for the UK used only the inclusive term of "serious adverse events" not further classified into YEL-And or YEL-AND and reported 34 "serious adverse events." The Swiss database used the term "serious adverse events" and reported 7 such events (including 4 "neurologic reactions") for a reporting rate of 25 "serious adverse events"/million doses. CONCLUSIONS: Reporting rates for serious adverse events following yellow fever vaccination are low. Differences in reporting rates may be due to differences in definitions, surveillance system organisation, methods of reporting cases, administration of YFV with other vaccines, incomplete information about denominators, time intervals for reporting events, the degree of passive reporting, access to diagnostic resources, and differences in time periods of reporting.

Active and passive surveillance of yellow fever vaccine 17D or 17DD-associated serious adverse events: systematic review.

PURPOSE: To identify the rate of serious adverse events attributable to yellow fever vaccination with 17D and 17DD strains reported in active and passive surveillance data. METHODS: We conducted a systematic review of published literature on adverse events associated with yellow fever. We searched 9 electronic databases for peer reviewed and grey literature in all languages. There were no restrictions on date of publication. Reference lists of key studies were also reviewed to identify additional studies. PRINCIPAL RESULTS: We identified 66 relevant studies: 24 used active, 17 a combination of passive and active (15 of which were pharmacovigilance databases), and 25 passive surveillance. ACTIVE SURVEILLANCE: A total of 2,660,929 patients in general populations were followed for adverse events after vaccination, heavily weighted (97.7%) by one large Brazilian study. There were no observed cases of viscerotropic or neurotropic disease, one of anaphylaxis and 26 cases of urticaria (hypersensitivity). We also identified four studies of infants and children (n=2199), four studies of women (n=1334), and one study of 174 HIV+, and no serious adverse events were observed. PHARMACOVIGILANCE DATABASES: 10 of the 15 databases contributed data to this review, with 107,621,154 patients, heavily weighted (94%) by the Brazilian database. The estimates for Australia were low at 0/210,656 for "severe neurological disease" and 1/210,656 for YEL-AVD, and also low for Brazil with 9 hypersensitivity events, 0.23 anaphylactic shock events, 0.84 neurologic syndrome events and 0.19 viscerotropic events cases/million doses. The five analyses of partly overlapping periods for the US VAERS database provided an estimate of 6.6 YEL-AVD and YEL-AND cases per million, and estimates between 11.1 and 15.6 of overall "serious adverse events" per million. The estimates for the UK were higher at 34 "serious adverse events" and also for Switzerland with 14.6 "neurologic events" and 40 "serious events not neurological"/million doses. PASSIVE SURVEILLANCE: Six studies of campaigns in general populations included 94,500,528 individuals, very heavily weighted (99%) by the Brazilian data, and providing an estimate of 0.51 serious AEFIs/million doses. Five retrospective reviews of hospital or clinic records included 60,698 individuals, and no serious AEFIs were proven. The data are heavily weighted (96%) by the data from the Hospital for Tropical Diseases, London. Two studies included 35,723 children, four studies included 138 pregnant women, six studies included 191 HIV+ patients, and there was one review of patients who were HIV+, and no serious AEFIs were proven. MAJOR CONCLUSIONS: The databases in each country used different definitions, protocols, surveillance mechanisms for the initial identification and reporting of cases, and strategies for the clinical and laboratory follow up of cases. The pharmacovigilance databases provide three sets of estimates: a low estimate from the Brazilian and Australian data, a medium estimate from the US VAERS data, and a higher estimate from the UK and Swiss data. The estimates from the active surveillance data are lower (and strongly influenced by the Brazilian data) and the estimates from the passive surveillance studies are also lower (strongly influenced by the London Hospital for Tropical Diseases data from the early 1950s). Sophisticated pathology, histopathology and tests such as PCR amplicon sequencing are needed to prove that serious adverse events were actually caused by the yellow fever vaccine, and the availability of such diagnostic capability is strongly biased towards recent reports from developed countries. Despite these variations in the estimation of serious harm, overall the 17D and 17DD yellow fever vaccine has proven to be a very safe vaccine and is highly effective against an illness with high potential mortality rates.

Molecular characterization of dermal lymphatic endothelial cells from primary lymphedema skin.

BACKGROUND: Lymphatic endothelial cells from primary lymphedema skin have never been cultured nor characterized. A subgroup of patients with primary lymphedema undergo surgery to bring about an improvement in their quality of life. The aim of this study was to culture and characterize LECs from the skin of these patients. METHODS AND RESULTS: Lymphatic endothelial cells were isolated and cultured from the skin of patients with primary lymphedema and from normal skin. The isolated cells were compared in their ability to form microvascular networks in a three-dimensional culture medium, and in their response to treatment with vascular endothelial growth factors A, C, and D. Whole tissue transcriptional profiling was carried out on two pools of isolated lymphatic endothelial cells--one from primary lymphedema skin and the other from normal skin. Lymphatic endothelial cells from primary lymphedema skin form tubule-like structures when cultured in three-dimensional media. They respond in a similar fashion to stimulation with the vascular endothelial growth factors A, C, and D. Comparative analysis between lymphedema tissue and normal tissue (fold change >2) showed differential expression of 2793 genes (5% of all transcripts), 2184 upregulated, and 609 downregulated. Genes involved in cellular apoptosis (vascular endothelial growth inhibitor, zinc finger protein), extracellular matrix turnover (matrix metalloproteinase inhibitor-16), and type IV collagen deposition were upregulated. Various pro-inflammatory genes (interleukin-6, interleukin-8, interleukin-32, E-selectin) were downregulated. CONCLUSION: Cellular adhesion, apoptosis, and increased extracellular matrix turnover play a more prominent role in primary lymphedema than previously thought. In addition, the acute inflammatory response is attenuated as evidenced by the downregulation of various pro-inflammatory genes.This sheds further light on the interplay of the various pathological processes taking place in primary lymphedema.

Phenotype and mRNA expression of syncytiotrophoblast microparticles isolated from human placenta.

Fetal and neonatal alloimmune thromboctyopenia due to maternal human platelet antigen (HPA)-1a antibodies affects primigravidas. Immunization must occur early in pregnancy before fetal platelets enter maternal blood via fetomaternal hemorrhage. The HPA-1a antigen is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin), which is also present on the placental syncytiotrophoblast (ST) and in direct contact with maternal blood. Since ST debris is shed into maternal blood during pregnancy, this material might be immunogenic in vivo. For experimental purposes, we prepared and characterized ST microparticles (STMPs) in vitro from term placentas. Phenotype analysis by flow cytometry and Western blotting showed that STMP expressed more placental alkaline phosphatase (PLAP) than GPIIIa. Quantitative real-time PCR demonstrated expression of human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and GPIIIa by STMP, in the order HPL > HCG > GPIIIa. PLAP, HPL, and HCG are trophoblast-specific proteins. These STMPs may be a useful model for studying the natural ST debris in plasma of pregnant women.