Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 10³ PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.     

The Gap between Individual Perception and Compliance: A Qualitative Follow-Up Study of the Surgical Safety Checklist Application

Background

“The Surgical Safety Checklist (SSC) is important, but we don’t use it adequately” is a well-suited statement that reflects the SSC''s application in hospitals. Our aim was to follow up on our initial study on compliance (2014) by analysing differences between individual perception and compliance with the SSC.

Methods

We conducted a follow-up online survey to assess healthcare professionals’ individual perception of, as well as satisfaction and compliance with the SSC three years following its thorough implementation.

Results

171 (19.5%) of 875 operating team members completed the online survey. 99.4% confirmed using the SSC. Self-estimated subjective knowledge about the intention of the checklist was high, whereas objective knowledge was moderate, but improved as compared to 2014. According to an independent audit the SSC was used in 93.1% of all operations and among the SSCs used the completion rate was 57.2%. The use of the SSC was rated as rather easy [median (IQR): 7 (6–7)], familiar [7 (6–7)], generally important [7 (7–7)], and good for patients [7 (6–7)] as well as for employees [7 (7–7)]. Only comfort of use was rated lower [6 (5–7)].

Conclusion

There is a gap between individual perception and actual application of the SSC. Despite healthcare professionals confirming the importance of the SSC, compliance was moderate. The introduction of SSCs in the health care sector remains a constant challenge and requires continuous re-evaluation as well as a sensible integration into existing workflows in hospitals.     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Exploring the diversity of marine-derived fungal polyketide synthases

Using an approach based on polymerase chain reaction (PCR), we examined the diversity of polyketide synthase (PKS) genes present in 160 marine fungal isolates, representing 142 species. We obtained ketosynthase (KS) domain PCR products from 99 fungal isolates, representing Dothideomycetes, Sordariomycetes, Eurotiomycetes, and incertae sedis. Sequence similarity searches and phylogenetic analysis of 29 marine partial-KS-encoding sequences revealed domains predicted to encode reducing, nonreducing, and 6-methylsalicylic acid PKSs. Bioinformatic analysis of an alignment of the KS sequences from marine-derived fungi revealed no unique motifs in this region. However, several specificity-determining positions were apparent between fungal 6-methylsalicylic acid PKSs as compared with either reducing or nonreducing PKSs. Evaluation of these positions in the context of a modelled three-dimensional protein structure highlighted their potential use as PKS classification markers. Evaluating primer-binding sites was necessary to obtain KS domain fragments from putative PKSs while maintaining a level of sequence information adequate to properly classify and characterize them.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.     

Diversity in mating behavior of hermaphroditic and male-female Caenorhabditis nematodes

In this study, we addressed why Caenorhabditis elegans males are inefficient at fertilizing their hermaphrodites. During copulation, hermaphrodites generally move away from males before they become impregnated. C. elegans hermaphrodites reproduce by internal self-fertilization, so that copulation with males is not required for species propagation. The hermaphroditic mode of reproduction could potentially relax selection for genes that optimize male mating behavior. We examined males from hermaphroditic and gonochoristic (male-female copulation) Caenorhabditis species to determine if they use different sensory and motor mechanisms to control their mating behavior. Instead, we found through laser ablation analysis and behavioral observations that hermaphroditic C. briggsae and gonochoristic C. remanei and Caenorhabditis species 4, PB2801 males produce a factor that immobilizes females during copulation. This factor also stimulates the vulval slit to widen, so that the male copulatory spicules can easily insert. C. elegans and C. briggsae hermaphrodites are not affected by this factor. We suggest that sensory and motor execution of mating behavior have not significantly changed among males of different Caenorhabditis species; however, during the evolution of internal self-fertilization, hermaphrodites have lost the ability to respond to the male soporific-inducing factor.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.