Outer membrane protein P5 of nontypeable (acapsulate)
Haemophilus influenzae (NTHi P5) forms large pores in planar lipid bilayers between symmetric solutions that unpredictably display a nonzero ...reversal potential. Moreover, NTHi P5 has a high theoretical isoelectric point, calculated as 9.58, which is not in agreement with the experimental isoelectric point, determined as 6.3–6.8, or with its preference for cations, disproportionately strong at one side. These anomalous results intimate that NTHi P5 is associated with a polyanion. Chemical and immunological analyses revealed the presence of inorganic polyphosphate (polyP), and the amphiphilic, solvating polyester, poly-(R)-3-hydroxybutyrate, frequently associated with polyP. A sharp reduction in cation selectivity was observed after addition of
Saccharomyces cerevisiae exopolyphosphatase X to the bilayer, providing functional evidence for the involvement of polyP in selectivity. The results suggest that NTHi P5 associates with polyP and poly-(R)-3-hydroxybutyrate to create large, cation-selective pores in the outer membrane of
H. influenzae.
Background Long-read sequencing is increasingly used to uncover structural variants in the human genome, both functionally neutral and deleterious. Structural variants occur more frequently in ...regions with a high homology or repetitive segments, and one rearrangement may predispose to additional events. Bartter syndrome type 3 (BS 3) is a monogenic tubulopathy caused by deleterious variants in the chloride channel gene CLCNKB, a high proportion of these being large gene deletions. Multiplex ligation-dependent probe amplification, the current diagnostic gold standard for this type of mutation, will indicate a simple homozygous gene deletion in biallelic deletion carriers. However, since the phenotypic spectrum of BS 3 is broad even among biallelic deletion carriers, we undertook a more detailed analysis of precise breakpoint regions and genomic structure. Methods Structural variants in 32 BS 3 patients from 29 families and one BS4b patient with CLCNKB deletions were investigated using long-read and synthetic long-read sequencing, as well as targeted long-read sequencing approaches. Results We report a ~3 kb duplication of 3'-UTR CLCNKB material transposed to the corresponding locus of the neighbouring CLCNKA gene, also found on ~50 % of alleles in healthy control individuals. This previously unknown common haplotype is significantly enriched in our cohort of patients with CLCNKB deletions (45 of 51 alleles with haplotype information, 2.2 kb and 3.0 kb transposition taken together, p=9.16x10.sup.-9). Breakpoint coordinates for the CLCNKB deletion were identifiable in 28 patients, with three being compound heterozygous. In total, eight different alleles were found, one of them a complex rearrangement with three breakpoint regions. Two patients had different CLCNKA/CLCNKB hybrid genes encoding a predicted CLCNKA/CLCNKB hybrid protein with likely residual function. Conclusions The presence of multiple different deletion alleles in our cohort suggests that large CLCNKB gene deletions originated from many independently recurring genomic events clustered in a few hot spots. The uncovered associated sequence transposition haplotype apparently predisposes to these additional events. The spectrum of CLCNKB deletion alleles is broader than expected and likely still incomplete, but represents an obvious candidate for future genotype/phenotype association studies. We suggest a sensitive and cost-efficient approach, consisting of indirect sequence capture and long-read sequencing, to analyse disease-relevant structural variant hotspots in general. Keywords: Bartter syndrome type 3, Salt-wasting tubulopathy, Long-read sequencing, Target enrichment, CLCNKA, CLCNKB, Structural variant, Risk haplotype, Next-generation sequencing, HiFi-sequencing
•Biopharmaceuticals are very promising for the treatment of multiple diseases.•Therapeutic proteins often bear sugar chains that can influence their pharmacology.•Glycosylation has to be ...characterized and controlled throughout drug development.•Mass spectrometry is a key technology in analysis of drug glycosylation.•Recent trends in mass spectrometry analysis of therapeutic glycoproteins.
Biopharmaceuticals are drugs of biotechnological origin used as vaccines or for the treatment of non-communicable diseases, such as cancer or anemia. Due to their high efficacy and specificity, the market for novel and biosimilar biopharmaceuticals is growing immensely. This growth is accompanied by new challenges in quality control and analytical characterization during drug development and production. Glycosylation is one of the structural modifications that occur during production of many protein-based drugs and can have significant effects on their pharmacological properties. Mass spectrometry (MS) is a promising technique for high-quality analytical characterization of glycosylation, starting from early drug development through to final lot release. Here, we review the most recent trends in biopharmaceutical glycosylation analysis by MS with and without coupling to liquid chromatography or capillary electrophoresis, and draw comparisons with established, non-MS methods. We discuss future prospects for the emerging MS approaches for the biotech industry and biopharmaceutical research.
Helminth parasites of teleost fish have evolved strategies to evade and manipulate the immune responses of their hosts. Responsiveness of fish host immunity to helminth antigens may therefore vary ...depending on the degree of host-parasite counter-adaptation. Generalist parasites, infective for a number of host species, might be unable to adapt optimally to the immune system of a certain host species, while specialist parasites might display high levels of adaptation to a particular host species. The degree of adaptations may further differ between sympatric and allopatric host-parasite combinations. Here, we test these hypotheses by in vitro exposure of head kidney leukocytes from three-spined sticklebacks (Gasterosteus aculeatus) to antigens from parasites with a broad fish host range (Diplostomum pseudospathaceum, Triaenophorus nodulosus), a specific fish parasite of cyprinids (Ligula intestinalis) and parasites highly specific only to a single fish species as second intermediate host (Schistocephalus pungitii, which does not infect G. aculeatus, and Schistocephalus solidus, infecting G. aculeatus). In vitro responses of stickleback leukocytes to S. solidus antigens from six European populations, with S. solidus prevalence from <1% to 66% were tested in a fully crossed experimental design. Leukocyte cultures were analysed by means of flow cytometry and a chemiluminescence assay to quantify respiratory burst activity. We detected decreasing magnitudes of in vitro responses to antigens from generalist to specialist parasites and among specialists, from parasites that do not infect G. aculeatus to a G. aculeatus-infecting species. Generalist parasites seem to maintain their ability to infect different host species at the costs of relatively higher immunogenicity compared to specialist parasites. In a comparison of sympatric and allopatric combinations of stickleback leukocytes and antigens from S. solidus, magnitudes of in vitro responses were dependent on the prevalence of the parasite in the population of origin, rather than on sympatry. Antigens from Norwegian (prevalence 30–50%) and Spanish (40–66%) S. solidus induced generally higher in vitro responses compared to S. solidus from two German (<1%) populations. Likewise, leukocytes from stickleback populations with a high S. solidus prevalence showed higher in vitro responses to S. solidus antigens compared to populations with low S. solidus prevalence. This suggests a rather low degree of local adaptation in S. solidus populations, which might be due to high gene flow among populations because of their extremely mobile final hosts, fish-eating birds.
•We investigated in vitro responses of stickleback leukocytes to parasite antigens.•In vitro responses decreased from generalist to specialist parasites.•Responses decreased from Gasterosteus aculeatus not infecting to infecting parasites.•Leukocyte response was increased with parasite prevalence in the natural habitat.•Immunogenicity of antigens was increased with parasite prevalence as well.
The nature of many microbe–host interactions is not static, but may shift along a continuum from mutualistic to harmful depending on the environmental conditions. In this study, we assessed the ...interaction between the foundation plant eelgrass Zostera marina and the frequently associated protist Labyrinthula zosterae. We tested how an important environmental factor, nutrient availability, would modulate their interaction. We experimentally infected naive eelgrass plants in combination with 2 nutrient levels (fertilized and non-fertilized). We followed L. zosterae infection, eelgrass growth parameters and host defense gene expression over 3 wk in large 600 l tanks. Inoculation with L. zosterae and nutrient limitation both reduced eelgrass growth. These effects were additive, whereas no interaction of nutrient treatment and L. zosterae inoculation was detected. Gene expression levels of 15 candidate genes revealed a reduced expression of photosynthesis-related genes but an increased expression of classical stress genes such as Hsp80 in inoculated plants 2 d post-inoculation. However, we found no effects on plant mortality, and plants were able to clear high infection levels within 3 wk to ambient background levels of infection as assessed via specific RT-qPCR designed to quantify endophytic L. zosterae. Thus, we found no evidence that L. zosterae is a facultative mutualist that facilitates eelgrass growth under nutrient-limiting conditions. We suggest that the interaction between contemporary L. zosterae genotypes and Z. marina represents a mild form of parasitism in northern Europe because the damage to the plant is moderate even under nutrient limitation stress.
Land cover transformations have accompanied the rise and fall of civilizations for thousands of years, exerting strong influence on the surrounding environment. Soil erosion and the associated ...outwash of nutrients are a main cause of eutrophication of aquatic ecosystems. Despite the great challenges of water protection in the face of climate change, large uncertainties remain concerning the timescales for recovery of aquatic ecosystems impacted by hypoxia. This study seeks to address this issue by investigating the sedimentary record of Lake Murten (Switzerland), which witnessed several phases of intensive human land-use over the past 2000 years.
Application of geophysical and geochemical methods to a 10 m-long sediment core revealed that soil erosion increased drastically with the rise of the Roman City of Aventicum (30 CE). During this period, the radiocarbon age of the bulk sedimentary organic carbon (OC) increasingly deviated from the modeled deposition age, indicating rapid flushing of old soil OC from the surrounding catchment driven by intensive land-use. Enhanced nutrient delivery resulted in an episode of cultural eutrophication, as shown by the deposition of varved sediments. Human activity drastically decreased towards the end of the Roman period (3rd century CE), resulting in land abandonment and renaturation. Recovery of the lake ecosystem from bottom-water hypoxia after the peak in human activity took around 50 years, while approximately 300 years passed until sediment accumulation reached steady state conditions on the surrounding landscape. These findings suggest that the legacy of anthropogenic perturbation to watersheds may persist for centuries.
•Radiocarbon anomalies as indicator for soil erosion.•A dramatic cultural eutrophication during the Roman period.•Long recovery time from eutrophication during renaturation phase.
The aim of this study was to evaluate the course of urethral obstruction in cats. Forty-five male cats with urethral obstruction or lower urinary tract signs referable to urethral obstruction were ...included in the study. Follow-up information was gained by telephone interview in most cases and was available in 39 cats. Of the 22 cats with idiopathic urethral obstruction, eight (36%) re-obstructed after 3–728 days (median 17 days). Of 10 cats with urolithiasis, three (30%) re-obstructed after 10, 13 and 472 days, respectively. Of the seven cats with urethral plugs, three (43%) re-obstructed after 4, 34 and 211 days, respectively. Recurrent signs of lower urinary tract disease including obstruction were common in cats with urethral obstruction (20/39; 51%) and occurred in the same frequency irrespective of the primary cause of the obstruction. Recurrent obstruction (14/39; 36%) was the most common reason for euthanasia and was performed in 8/39 (21%) cats.
T cell immunity is central for the control of viral infections. CoVac-1 is a peptide-based vaccine candidate, composed of SARS-CoV-2 T cell epitopes derived from various viral proteins
, combined ...with the Toll-like receptor 1/2 agonist XS15 emulsified in Montanide ISA51 VG, aiming to induce profound SARS-CoV-2 T cell immunity to combat COVID-19. Here we conducted a phase I open-label trial, recruiting 36 participants aged 18-80 years, who received a single subcutaneous CoVac-1 vaccination. The primary end point was safety analysed until day 56. Immunogenicity in terms of CoVac-1-induced T cell response was analysed as the main secondary end point until day 28 and in the follow-up until month 3. No serious adverse events and no grade 4 adverse events were observed. Expected local granuloma formation was observed in all study participants, whereas systemic reactogenicity was absent or mild. SARS-CoV-2-specific T cell responses targeting multiple vaccine peptides were induced in all study participants, mediated by multifunctional T helper 1 CD4
and CD8
T cells. CoVac-1-induced IFNγ T cell responses persisted in the follow-up analyses and surpassed those detected after SARS-CoV-2 infection as well as after vaccination with approved vaccines. Furthermore, vaccine-induced T cell responses were unaffected by current SARS-CoV-2 variants of concern. Together, CoVac-1 showed a favourable safety profile and induced broad, potent and variant of concern-independent T cell responses, supporting the presently ongoing evaluation in a phase II trial for patients with B cell or antibody deficiency.
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