Mim8, a novel factor VIIIa mimetic bispecific antibody, shows favorable safety and pharmacokinetics in healthy adults.

Background: Mim8 (denecimig) is a novel activated coagulation factor VIII-mimetic bispecific antibody that assembles with activated coagulation FIX and FX on the platelet membrane surface. Objectives: The FRONTIER1 (NCT04204408, NN7769-4513) single ascending dose and the 4882 pharmacokinetic (PK) studies (NCT05127473, NN7769-4882) examined the safety, tolerability, PK, and pharmacodynamics (PD) of Mim8 in healthy adult males. Methods: The FRONTIER1 single ascending dose study consisted of 6 cohorts, each with 6 participants who received a single subcutaneous (s.c.) dose of Mim8 and 2 participants who received a placebo. The 4882 PK study had 11 arms, each with 6 participants who received a single s.c. dose of Mim8. The primary endpoint for both studies was treatment-emergent adverse events. Other safety assessments included relative changes in D-dimer, prothrombin fragments 1 and 2, fibrinogen, and platelets. The PK and PD were assessed using Mim8 plasma concentration and activated partial thromboplastin clotting time and thrombin generation, respectively. Results: Mim8 was well tolerated, and there were no severe treatment-emergent adverse events. The PK properties of Mim8 in both studies were consistent with dose-proportionality. The terminal half-life of Mim8 after a single dose was approximately 1 month, and maximum plasma concentration was reached after 10 days. Conclusion: The PK and PD profiles suggest that Mim8 is suitable as a long-acting FVIIIa-mimetic bispecific antibody for hemophilia A prophylaxis.

Temporal and spatial dynamics of Fusarium spp. and mycotoxins in Swedish cereals during 16 years.

We analysed the dynamics of Fusarium spp. and mycotoxin contamination in Swedish cereals during 2004-2018. More than 1400 cereal samples from field trials were included, collected in a monitoring programme run by the Swedish Board of Agriculture. Five Fusarium mycotoxins were quantified with LC-MS/MS and fungal DNA from four species was quantified using quantitative real-time PCR. Correlation analyses revealed that deoxynivalenol (DON) and zearalenone (ZEN) were mainly associated with Fusarium graminearum, but stronger correlations with F. culmorum was seen some years. Nivalenol (NIV) was associated with F. poae and the HT-2 and T-2 toxins with F. langsethiae. Clear differences in mycotoxin contamination between different cereal crops and geographical regions were identified. The highest levels of DON and ZEN were found in spring wheat in Western Sweden. For NIV, HT-2 and T-2 toxins, the levels were highest in spring oats and spring barley. Regional differences were not detected for NIV, while HT-2 and T-2 toxins were associated with the northernmost region. We found that delayed harvest was strongly associated with increased levels of DON and ZEN in several crops. However, harvest date did not influence the levels of NIV or HT-2 and T-2 toxins. Our results suggest similar distribution patterns of DON and ZEN, in contrast to NIV and HT-2 and T-2 toxins, probably mirroring the differences in the ecology of the toxin-producing Fusarium species. Timely harvest is important to reduce the risk of DON and ZEN contamination, especially for fields with other risk factors.

Fusarium Head Blight From a Microbiome Perspective.

The fungal genus Fusarium causes several diseases in cereals, including Fusarium head blight (FHB). A number of Fusarium species are involved in disease development and mycotoxin contamination. Lately, the importance of interactions between plant pathogens and the plant microbiome has been increasingly recognized. In this review, we address the significance of the cereal microbiome for the development of Fusarium-related diseases. Fusarium fungi may interact with the host microbiome at multiple stages during their life cycles and in different plant organs including roots, stems, leaves, heads, and crop residues. There are interactions between Fusarium and other fungi and bacteria as well as among Fusarium species. Recent studies have provided a map of the cereal microbiome and revealed how different biotic and abiotic factors drive microbiome assembly. This review synthesizes the current understanding of the cereal microbiome and the implications for Fusarium infection, FHB development, disease control, and mycotoxin contamination. Although annual and regional variations in predominant species are significant, much research has focused on Fusarium graminearum. Surveying the total Fusarium community in environmental samples is now facilitated with novel metabarcoding methods. Further, infection with multiple Fusarium species has been shown to affect disease severity and mycotoxin contamination. A better mechanistic understanding of such multiple infections is necessary to be able to predict the outcome in terms of disease development and mycotoxin production. The knowledge on the composition of the cereal microbiome under different environmental and agricultural conditions is growing. Future studies are needed to clearly link microbiome structure to Fusarium suppression in order to develop novel disease management strategies for example based on conservation biological control approaches.

Body Mass Index Best Predicts Recovery of Recombinant Factor VIII in Underweight to Obese Patients with Severe Haemophilia A.

BACKGROUND: Factor VIII (FVIII) products are usually dosed according to body weight (BW). This may lead to under- or over-dosing in underweight or obese patients, respectively. OBJECTIVE: This article evaluates the pharmacokinetics (PK) of recombinant FVIII concentrate, particularly recovery, in relation to body mass index (BMI) and other body composition descriptors. MATERIALS AND METHODS: Thirty-five previously treated adults with severe haemophilia A from five BMI categories (underweight, normal, overweight, obese class I and II/III) were included. PK was evaluated after 50 IU per kilogram of BW single-dose recombinant FVIII (turoctocog alfa). The body composition variable was based on measurements of weight, height, bioimpedance analysis, and dual-energy X-ray absorptiometry. A dosing model was derived to achieve similar peak FVIII activity levels across BMI categories. RESULTS: A statistically significant positive association between BMI and C30min, IR30min, and AUC0-inf was observed; CL and Vss showed a significant negative association with BMI; t½ was independent of BMI and other parameters. The dosing model introduced a correction factor 'M' for each BMI category, based on linear regression analysis of C30min against BMI, which ranged from 0.55 for underweight to 0.39 for obese class II/III. This model achieved similar peak FVIII activity levels across BMI categories, estimating an average dose adjustment of +243.3 IU (underweight) to -1,489.6 IU (obese class II/III) to achieve similar C30min. CONCLUSION: BMI appears to be the best predictor of recombinant FVIII recovery; however, PK endpoints were also dependent on other body composition variables. The model demonstrated that dosing can be adjusted for individual BMI to achieve better FVIII predictability across BMI categories.

Preceding crop and tillage system affect winter survival of wheat and the fungal communities on young wheat roots and in soil.

Agricultural practices like tillage and cropping sequence have profound influence on soil-living and plant-associated fungi, and thereby on plant growth. In a field experiment, we studied the effects of preceding crop and tillage on fungal communities in the soil and on young winter wheat roots in relation to plant winter survival and grain yield. We hypothesized that plant performance and fungal communities (described by amplicon sequencing) differ depending on tillage system and preceding crop; that the effect of preceding crop differs depending on tillage system, and that differences in fungal communities are reflected in plant performance. In line with our hypotheses, effects of preceding crop on plant growth and fungal communities on plant roots and in soil were more pronounced under non-inversion tillage than under inversion tillage (ploughing). Fungal communities on plant roots in treatments with low winter survival were different from those with better survival. In soil, several fungal OTUs (operational taxonomic units) differed significantly between tillage systems. OTUs representing putative plant pathogens were either more abundant (Parastagonospora sp._27) or less abundant (Fusarium culmorum/graminearum_5) after non-inversion tillage. Our findings highlight the influence of cultural practices on fungal communities and thereby on plant health and yield.

Favorable pharmacokinetics in hemophilia B for nonacog beta pegol versus recombinant factor IX-Fc fusion protein: A randomized trial.

BACKGROUND AND OBJECTIVE: Nonacog beta pegol (N9-GP) and recombinant factor IX-Fc fusion protein (rFIXFc) are extended half-life rFIX compounds. We report the first single-dose pharmacokinetic trial of N9-GP and rFIXFc. PATIENTS/METHODS: Paradigm 7 was a multicenter, open-label, randomized, crossover trial in previously treated (>150 exposure days) adults with congenital hemophilia B (FIX activity ≤2%). Patients received single intravenous injections (50 IU/kg) of N9-GP and rFIXFc with at least 21 days between doses. Plasma FIX activity, predose, and at serial time points up to 240 hours postdose, was measured using validated one-stage clotting assays (SynthAFax for N9-GP; Actin FSL for rFIXFc) and a chromogenic assay (ROX factor IX) with normal human plasma as calibrator. The primary endpoint was area under the FIX activity-time curve from 0 to infinity, dose-normalized to 50 IU/kg (AUC0-inf,norm). RESULTS: Fifteen patients received study treatment. Based on FIX activity results from the one-stage clotting assays, estimated AUC0-inf,norm was significantly greater for N9-GP than rFIXFc (ratio: 4.39; P < 0.0001, based on a two-sided test on 5% significance level). In addition, N9-GP had a longer terminal half-life, two times higher incremental recovery at 30 minutes and maximum FIX activity (dose-normalized to 50 IU/kg) and six times higher FIX activity at 168 hours than rFIXFc. These findings were largely comparable with the chromogenic assay data and are consistent with published data for each compound. CONCLUSIONS: In this comparison, N9-GP demonstrated favorable pharmacokinetic characteristics versus rFIXFc, helping clinicians to understand differences between N9-GP and rFIXFc. REGISTRATION: This trial is registered with clinicaltrials.gov (NCT03075670) and the European Clinical Trials Database (EudraCT: 2016-001149-25).

Agricultural factors affecting Fusarium communities in wheat kernels.

Fusarium head blight (FHB) is a devastating disease of cereals caused by Fusarium fungi. The disease is of great economic importance especially owing to reduced grain quality due to contamination by a range of mycotoxins produced by Fusarium. Disease control and prediction is difficult because of the many Fusarium species associated with FHB. Different species may respond differently to control methods and can have both competitive and synergistic interactions. Therefore, it is important to understand how agricultural practices affect Fusarium at the community level. Lower levels of Fusarium mycotoxin contamination of organically produced cereals compared with conventionally produced have been reported, but the causes of these differences are not well understood. The aim of our study was to investigate the effect of agricultural factors on Fusarium abundance and community composition in different cropping systems. Winter wheat kernels were collected from 18 organically and conventionally cultivated fields in Sweden, paired based on their geographical distance and the wheat cultivar grown. We characterised the Fusarium community in harvested wheat kernels using 454 sequencing of translation elongation factor 1-α amplicons. In addition, we quantified Fusarium spp. using real-time PCR to reveal differences in biomass between fields. We identified 12 Fusarium operational taxonomic units (OTUs) with a median of 4.5 OTUs per field. Fusarium graminearum was the most abundant species, while F. avenaceum had the highest occurrence. The abundance of Fusarium spp. ranged two orders of magnitude between fields. Two pairs of Fusarium species co-occurred between fields: F. poae with F. tricinctum and F. culmorum with F. sporotrichoides. We could not detect any difference in Fusarium communities between the organic and conventional systems. However, agricultural intensity, measured as the number of pesticide applications and the amount of nitrogen fertiliser applied, had an impact on Fusarium communities, specifically increasing the abundance of F. tricinctum. There were geographical differences in the Fusarium community composition where F. graminearum was more abundant in the western part of Sweden. The application of amplicon sequencing provided a comprehensive view of the Fusarium community in cereals. This gives us better opportunities to understand the ecology of Fusarium spp., which is important in order to limit FHB and mycotoxin contamination in cereals.

Organic farming increases richness of fungal taxa in the wheat phyllosphere.

Organic farming is often advocated as an approach to mitigate biodiversity loss on agricultural land. The phyllosphere provides a habitat for diverse fungal communities that are important for plant health and productivity. However, it is still unknown how organic farming affects the diversity of phyllosphere fungi in major crops. We sampled wheat leaves from 22 organically and conventionally cultivated fields in Sweden, paired based on their geographical location and wheat cultivar. Fungal communities were described using amplicon sequencing and real-time PCR. Species richness was higher on wheat leaves from organically managed fields, with a mean of 54 operational taxonomic units (OTUs) compared with 40 OTUs for conventionally managed fields. The main components of the fungal community were similar throughout the 350-km-long sampling area, and seven OTUs were present in all fields: Zymoseptoria, Dioszegia fristingensis, Cladosporium, Dioszegia hungarica, Cryptococcus, Ascochyta and Dioszegia. Fungal abundance was highly variable between fields, 103 -105 internal transcribed spacer copies per ng wheat DNA, but did not differ between cropping systems. Further analyses showed that weed biomass was the strongest explanatory variable for fungal community composition and OTU richness. These findings help provide a more comprehensive understanding of the effect of organic farming on the diversity of organism groups in different habitats within the agroecosystem.

Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

Fungicide effects on fungal community composition in the wheat phyllosphere.

The fungicides used to control diseases in cereal production can have adverse effects on non-target fungi, with possible consequences for plant health and productivity. This study examined fungicide effects on fungal communities on winter wheat leaves in two areas of Sweden. High-throughput 454 sequencing of the fungal ITS2 region yielded 235 operational taxonomic units (OTUs) at the species level from the 18 fields studied. It was found that commonly used fungicides had moderate but significant effect on fungal community composition in the wheat phyllosphere. The relative abundance of several saprotrophs was altered by fungicide use, while the effect on common wheat pathogens was mixed. The fungal community on wheat leaves consisted mainly of basidiomycete yeasts, saprotrophic ascomycetes and plant pathogens. A core set of six fungal OTUs representing saprotrophic species was identified. These were present across all fields, although overall the difference in OTU richness was large between the two areas studied.

Concentration- and time-dependent effects of isothiocyanates produced from Brassicaceae shoot tissues on the pea root rot pathogen Aphanomyces euteiches.

Isothiocyanates (ITCs) hydrolyzed from glucosinolates (GSLs) in Brassicaceae tissue are toxic to soil organisms. In this study, the effect of aliphatic and aromatic ITCs from hydrated dry Brassicaceae shoot tissues on the mycelium and oospores of the pea root rot pathogen Aphanomyces euteiches was investigated. The profile and concentrations of GSLs in two test Brassicaceae species, Sinapis alba and Brassica juncea, and the ITCs from the dominant hydrolyzed parent GSLs were monitored. The concentrations of dominant ITCs and pathogen exposure time were evaluated in in vitro experiments. The greatest effect on the pathogen was observed from aliphatic ITCs hydrolyzed from B. juncea tissue, and the effect depended on the ITC concentration and exposure time. ITCs were more effectively hydrolyzed from B. juncea GSLs than from S. alba GSLs; i.e., the ITC/GSL ratio was higher in B. juncea than in S. alba tissue, giving a different release pattern. The release of phenylethyl isothiocyanate, which was common to both species, followed a pattern similar to that of the dominant ITC in each crop species. This suggests that traits other than GSL content, e.g., plant cell structure, may affect the release of ITCs and should therefore influence the choice of species used for biofumigation purposes.

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores.

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.

Muscular exercise can cause highly pathological liver function tests in healthy men.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The occurrence of idiosyncratic drug hepatotoxicity is a major problem in all phases of clinical drug development and the leading cause of postmarketing warnings and withdrawals. Physical exercise can result in transient elevations of liver function tests. There is no consensus in the literature on which forms of exercise may cause changes in liver function tests and to what extent. WHAT THIS STUDY ADDS: Weightlifting results in profound increases in liver function tests in healthy men used to moderate physical activity, not including weightlifting. Liver function tests are significantly increased for at least 7 days after weightlifting. It is important to impose relevant restrictions on heavy muscular exercise prior to and during clinical studies. AIM: To investigate the effect of intensive muscular exercise (weightlifting) on clinical chemistry parameters reflecting liver function in healthy men. METHODS: Fifteen healthy men, used to moderate physical activity not including weightlifting, performed an 1 h long weightlifting programme. Blood was sampled for clinical chemistry parameters [aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LD), gamma-glutamyl transferase (gamma GT), alkaline phosphatase (ALP), bilirubin, creatine kinase (CK) and myoglobin] at repeated intervals during 7 days postexercise and at a follow-up examination 10-12 days postexercise. RESULTS: Five out of eight studied clinical chemistry parameters (AST, ALT, LD, CK and myoglobin) increased significantly after exercise (P < 0.01) and remained increased for at least 7 days postexercise. Bilirubin, gamma GT and ALP remained within the normal range. CONCLUSION: The liver function parameters, AST and ALT, were significantly increased for at least 7 days after the exercise. In addition, LD and, in particular, CK and myoglobin showed highly elevated levels. These findings highlight the importance of imposing restrictions on weightlifting prior to and during clinical studies. Intensive muscular exercise, e.g. weightlifting, should also be considered as a cause of asymptomatic elevations of liver function tests in daily clinical practice.

Ubiquilin-1 is a novel HASH-1-complexing protein that regulates levels of neuronal bHLH transcription factors in human neuroblastoma cells.

The basic helix-loop-helix (bHLH) transcription factor mammalian achaete-scute homologue-1 (MASH-1 in mouse and HASH-1 in humans) is expressed in specific subsets of embryonic neuronal precursors of both the peripheral and central nervous systems. This gene is essential for development of olfactory and most peripheral autonomic neurons. Neuro-blastoma is a pediatric malignancy derived from sympathetic nervous system precursors and HASH-1 is expressed in a majority of neuroblastoma tumors and cell lines, indicating the immature phenotype of these cells. Using a human neuroblastoma cDNA library and the yeast two-hybrid system to identify novel HASH-1-interacting proteins, we isolated ubiquilin-1 (DA41, hPLIC-1), a gene that contains multiple ubiquitin-related domains. Further analyses showed that ubiquilin-1 interacts not only with HASH-1, but also with other tissue-specific bHLH proteins, including HES-1. Overexpression of ubiquilin-1 led to accumulation of HASH-1 and HES-1. Moreover, ubiquilin-1 was translocated from the cytoplasm to the nucleus upon co-expression with HASH-1. These results indicate that ubiquilin-1 plays an active role in the precise regulation of HASH-1 and of other tissue-specific bHLH proteins.

Olf/EBF proteins are expressed in neuroblastoma cells: potential regulators of the Chromogranin A and SCG10 promoters.

The childhood malignancy neuroblastoma is derived from developmentally arrested sympathetic nervous system precursor cells. To obtain further insight into the molecular processes involved in the formation of these tumors, we decided to investigate the functional role of Olf/EBF (O/E) transcription factors in human neuroblastoma cells. We here report that O/E-1 and O/E-2 are expressed at variable levels in neuroblastoma cell lines and that O/E proteins could be identified by electrophoretic mobility shift assays. To identify potential neuronal target genes for O/E proteins in neuroblastoma cells we investigated the ability of a set of neuronal promoters to interact with O/E-1 in electrophoretic mobility shift assays. This analysis suggested that the Chromogranin A (CgA) and SCG10 promoters both contained binding sites for O/E-1. O/E-1 was able to activate the CgA promoter in vivo and mutation of the O/E-1 binding site in the CgA promoter reduced the functional activity of the element to about 60% of the wild-type in neuroblastoma cells, supporting the idea that O/E proteins may be involved in the control of the CgA promoter. Furthermore, overexpression of O/E-1 in hippocampal progenitor cells led to neurite outgrowth, indicative of a role for O/E proteins in neuronal differentiation.

A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum.

Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.

Modulation of basic helix-loop-helix transcription complex formation by Id proteins during neuronal differentiation.

It is assumed that the Id helix-loop-helix (HLH) proteins act by associating with ubiquitously expressed basic HLH (bHLH) transcription factors, such as E47 and E2-2, which prevents these factors from forming functional hetero- or homodimeric DNA binding complexes. Several tissue-specific bHLH proteins, including HASH-1, dHAND, and HES-1, are important for development of the nervous system. Neuroblastoma tumors are derived from the sympathetic nervous system and exhibit neural crest features. In differentiating neuroblastoma cells, HASH-1 is down-regulated, and there is coincident up-regulation of the transcriptional repressor HES-1, which is known to bind the HASH-1 promoter. We found that the three Id proteins expressed in neuroblastoma cells (Id1, Id2, and Id3) were down-regulated during induced differentiation, indicating that Id proteins help keep the tumor cells in an undifferentiated state. Studying interactions, we noted that all four Id proteins could dimerize with E47 or E2-2, but not with HASH-1 or dHAND. However, the Id proteins did complex with HES-1, and increased levels of Id2 reduced the DNA binding activity of HES-1. Furthermore, HES-1 interfered with Id2/E2-2 complex formation. The ability of Id proteins to affect HES-1 activity is of particular interest in neuronal cells, where regulation of HES-1 is essential for the timing of neuronal differentiation.