Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha.

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.

Correction to: An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola).

An amendment to this paper has been published and can be accessed via the original article.

An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola).

BACKGROUND: The latest advancements in DNA sequencing technologies have facilitated the resolution of the phylogeny of insects, yet parts of the tree of Holometabola remain unresolved. The phylogeny of Neuropterida has been extensively studied, but no strong consensus exists concerning the phylogenetic relationships within the order Neuroptera. Here, we assembled a novel transcriptomic dataset to address previously unresolved issues in the phylogeny of Neuropterida and to infer divergence times within the group. We tested the robustness of our phylogenetic estimates by comparing summary coalescent and concatenation-based phylogenetic approaches and by employing different quartet-based measures of phylogenomic incongruence, combined with data permutations. RESULTS: Our results suggest that the order Raphidioptera is sister to Neuroptera + Megaloptera. Coniopterygidae is inferred as sister to all remaining neuropteran families suggesting that larval cryptonephry could be a ground plan feature of Neuroptera. A clade that includes Nevrorthidae, Osmylidae, and Sisyridae (i.e. Osmyloidea) is inferred as sister to all other Neuroptera except Coniopterygidae, and Dilaridae is placed as sister to all remaining neuropteran families. Ithonidae is inferred as the sister group of monophyletic Myrmeleontiformia. The phylogenetic affinities of Chrysopidae and Hemerobiidae were dependent on the data type analyzed, and quartet-based analyses showed only weak support for the placement of Hemerobiidae as sister to Ithonidae + Myrmeleontiformia. Our molecular dating analyses suggest that most families of Neuropterida started to diversify in the Jurassic and our ancestral character state reconstructions suggest a primarily terrestrial environment of the larvae of Neuropterida and Neuroptera. CONCLUSION: Our extensive phylogenomic analyses consolidate several key aspects in the backbone phylogeny of Neuropterida, such as the basal placement of Coniopterygidae within Neuroptera and the monophyly of Osmyloidea. Furthermore, they provide new insights into the timing of diversification of Neuropterida. Despite the vast amount of analyzed molecular data, we found that certain nodes in the tree of Neuroptera are not robustly resolved. Therefore, we emphasize the importance of integrating the results of morphological analyses with those of sequence-based phylogenomics. We also suggest that comparative analyses of genomic meta-characters should be incorporated into future phylogenomic studies of Neuropterida.

Four myriapod relatives - but who are sisters? No end to debates on relationships among the four major myriapod subgroups.

BACKGROUND: Phylogenetic relationships among the myriapod subgroups Chilopoda, Diplopoda, Symphyla and Pauropoda are still not robustly resolved. The first phylogenomic study covering all subgroups resolved phylogenetic relationships congruently to morphological evidence but is in conflict with most previously published phylogenetic trees based on diverse molecular data. Outgroup choice and long-branch attraction effects were stated as possible explanations for these incongruencies. In this study, we addressed these issues by extending the myriapod and outgroup taxon sampling using transcriptome data. RESULTS: We generated new transcriptome data of 42 panarthropod species, including all four myriapod subgroups and additional outgroup taxa. Our taxon sampling was complemented by published transcriptome and genome data resulting in a supermatrix covering 59 species. We compiled two data sets, the first with a full coverage of genes per species (292 single-copy protein-coding genes), the second with a less stringent coverage (988 genes). We inferred phylogenetic relationships among myriapods using different data types, tree inference, and quartet computation approaches. Our results unambiguously support monophyletic Mandibulata and Myriapoda. Our analyses clearly showed that there is strong signal for a single unrooted topology, but a sensitivity of the position of the internal root on the choice of outgroups. However, we observe strong evidence for a clade Pauropoda+Symphyla, as well as for a clade Chilopoda+Diplopoda. CONCLUSIONS: Our best quartet topology is incongruent with current morphological phylogenies which were supported in another phylogenomic study. AU tests and quartet mapping reject the quartet topology congruent to trees inferred with morphological characters. Moreover, quartet mapping shows that confounding signal present in the data set is sufficient to explain the weak signal for the quartet topology derived from morphological characters. Although outgroup choice affects results, our study could narrow possible trees to derivatives of a single quartet topology. For highly disputed relationships, we propose to apply a series of tests (AU and quartet mapping), since results of such tests allow to narrow down possible relationships and to rule out confounding signal.

Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes.

Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC.IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.

Radical Reactions Induced by Visible Light in Dichloromethane Solutions of Hünig's Base: Synthetic Applications and Mechanistic Observations.

ß-(3-Iodopropoxy)-substituted α,ß-unsaturated lactams, lactones, and cycloalkenones (eight examples) underwent reductive radical reactions in a dichloromethane solution of N,N-diisopropylethylamine (Hünig's base) upon irradiation with visible light (λ=419 nm). Apart from plain reduction reactions (hydro-de-iodination), a significant degree of cyclization was observed in three cases. In parallel to the conversion of the substrates, the formation of intensely colored by-products was observed. Based on mass spectrometric evidence and upon comparison with known compounds, the by-products were identified as cyanine dyes. Their formation supports the hypothesis that irradiation of dichloromethane solutions of Hünig's base leads to the formation of radicals, which in turn can either initiate a radical reaction or combine with cyanine precursors. It was shown by deuterium-labelling experiments, that one equivalent of dichloromethane is incorporated into the cyanine dyes and that the reductive quenching of radical intermediates is at least partially due to hydrogen abstraction from the solvent. As a consequence, a reductive cyclization of the starting materials is favored in CD2 Cl2 solutions as shown for two ß-(3-iodopropoxy)-substituted tetronates, which underwent in dichloromethane almost exclusive reduction, but gave predominantly the cyclization products in CD2 Cl2 .

The ocelli of Archaeognatha (Hexapoda): functional morphology, pigment migration and chemical nature of the reflective tapetum.

The ocelli of Archaeognatha, or jumping bristletails, differ from typical insect ocelli in shape and field of view. Although the shape of the lateral ocelli is highly variable among species, most Machiloidea have sole-shaped lateral ocelli beneath the compound eyes and a median ocellus that is oriented downward. This study investigated morphological and physiological aspects of the ocelli of Machilis hrabei and Lepismachilis spp. The light-reflecting ocellar tapetum in M. hrabei is made up of xanthine nanocrystals, as demonstrated by confocal Raman spectroscopy. Pigment granules in the photoreceptor cells move behind the tapetum in the dark-adapted state. Such a vertical pigment migration in combination with a tapetum has not been described for any insect ocellus so far. The pigment migration has a dynamic range of approximately 4 log units and is maximally sensitive to green light. Adaptation from darkness to bright light lasts over an hour, which is slow compared with the radial pupil mechanism in some dragonflies and locusts.

A new kind of auxiliary heart in insects: functional morphology and neuronal control of the accessory pulsatile organs of the cricket ovipositor.

INTRODUCTION: In insects, the pumping of the dorsal heart causes circulation of hemolymph throughout the central body cavity, but not within the interior of long body appendages. Hemolymph exchange in these dead-end structures is accomplished by special flow-guiding structures and/or autonomous pulsatile organs ("auxiliary hearts"). In this paper accessory pulsatile organs for an insect ovipositor are described for the first time. We studied these organs in females of the cricket Acheta domesticus by analyzing their functional morphology, neuroanatomy and physiological control. RESULTS: The lumen of the four long ovipositor valves is subdivided by longitudinal septa of connective tissue into efferent and afferent hemolymph sinuses which are confluent distally. The countercurrent flow in these sinuses is effected by pulsatile organs which are located at the bases of the ovipositor valves. Each of the four organs consists of a pumping chamber which is compressed by rhythmically contracting muscles. The morphology of the paired organs is laterally mirrored, and there are differences in some details between the dorsal and ventral organs. The compression of the pumping chambers of each valve pair occurs with a left-right alternating rhythm with a frequency of 0.2 to 0.5 Hz and is synchronized between the dorsal and ventral organs. The more anteriorly located genital chamber shows rhythmical lateral movements simultaneous to those of the ovipositor pulsatile organs and probably supports the hemolymph exchange in the abdominal apex region. The left-right alternating rhythm is produced by a central pattern generator located in the terminal ganglion. It requires no sensory feedback for its output since it persists in the completely isolated ganglion. Rhythm-modulating and rhythm-resetting interneurons are identified in the terminal ganglion. CONCLUSION: The circulatory organs of the cricket ovipositor have a unique functional morphology. The pumping apparatus at the base of each ovipositor valve operates like a bellow. It forces hemolymph via sinuses delimited by thin septa of connective tissue in a countercurrent flow through the valve lumen. The pumping activity is based on neurogenic control by a central pattern generator in the terminal ganglion.

Unravelling the origins of boldness behaviour: a common garden experiment with cavefish (Barbatula barbatula).

Many animals show an aversion to bright, open spaces, with significant variability seen across species, populations and individuals within populations. Although there is much interest in the underlying causes of this behaviour, few studies have been able to systematically isolate the role of heritable and environmental effects. Here, we addressed this gap using a common garden experiment with cavefish. Specifically, we bred and cross-bred cave loaches (Barbatula barbatula), Europe's only known cavefish, in the laboratory, raised the offspring in complete darkness or normal light conditions, and studied their light avoidance behaviour. Cavefish spent much more time in a light area and ventured further out, while surface fish spent considerable time in risk-assessment behaviour between the light and dark areas. Hybrids behaved most similarly to cavefish. Light treatment and eye quality and lens size only had a modest effect. Our results suggest light avoidance behaviour of cavefish has a heritable basis and is fundamentally linked to increased boldness rather than reduced vision, which is likely adaptive given the complete lack of macropredators in the cave environment. Our study provides novel experimental insights into the behavioural divergence of cavefish and contributes to our broader understanding of the evolution of boldness and behavioural adaptation.

Genetic differentiation and phenotypic plasticity drive troglomorphic character development in European cavefish.

The Aach cave loach (Barbatula barbatula), a recently discovered member of the Nemacheilidae family, offers a unique opportunity to understand the mechanisms underlying evolutionary change. In a common garden experiment, we reared groups of laboratory-bred cave, surface, and hybrid loach under different light conditions. Troglomorphic characters varied significantly among the fish, influenced to a different extent by parental origin and light conditions. Cavefish progeny consistently exhibited smaller eyes, lighter pigmentation, longer barbels, and larger olfactory epithelia than surface fish, while hybrids displayed intermediate characteristics. Surface and hybrid fish raised in complete darkness resembled the cavefish phenotype, while cavefish raised under a natural photoperiod approached the surface form. Characters associated with eye degeneration were found to be primarily heritable. Conversely, traits related to chemo- and mechano-reception were enhanced in the surface and hybrid groups reared in complete darkness, suggesting phenotypic plasticity. Our findings offer valuable insights into the interplay between genetic differentiation and phenotypic plasticity to troglomorphic adaption. This contributes to the broader understanding of the early stages of adaptation, where phenotypic plasticity, drift, and selection shape phenotypes. Relatively recently established cavefish, such as the Aach cave loach, are promising candidates for comparative research investigating evolutionary mechanisms.

Adhesion of human and animal Escherichia coli strains in association with their virulence-associated genes and phylogenetic origins.

Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.

Brain anatomy in Diplura (Hexapoda).

BACKGROUND: In the past decade neuroanatomy has proved to be a valuable source of character systems that provide insights into arthropod relationships. Since the most detailed description of dipluran brain anatomy dates back to Hanström (1940) we re-investigated the brains of Campodea augens and Catajapyx aquilonaris with modern neuroanatomical techniques. The analyses are based on antibody staining and 3D reconstruction of the major neuropils and tracts from semi-thin section series. RESULTS: Remarkable features of the investigated dipluran brains are a large central body, which is organized in nine columns and three layers, and well developed mushroom bodies with calyces receiving input from spheroidal olfactory glomeruli in the deutocerebrum. Antibody staining against a catalytic subunit of protein kinase A (DC0) was used to further characterize the mushroom bodies. The japygid Catajapyx aquilonaris possesses mushroom bodies which are connected across the midline, a unique condition within hexapods. CONCLUSIONS: Mushroom body and central body structure shows a high correspondence between japygids and campodeids. Some unique features indicate that neuroanatomy further supports the monophyly of Diplura. In a broader phylogenetic context, however, the polarization of brain characters becomes ambiguous. The mushroom bodies and the central body of Diplura in several aspects resemble those of Dicondylia, suggesting homology. In contrast, Archaeognatha completely lack mushroom bodies and exhibit a central body organization reminiscent of certain malacostracan crustaceans. Several hypotheses of brain evolution at the base of the hexapod tree are discussed.

Reviving the Workshop Series on Testing Database Systems - DBTest.

With the ever increasing complexity of database systems and their pervasive use in industry, testing them has been an important issue for a long time. Recognizing this relevance, researchers and industry have started the Workshop Series on Testing Database Systems in 2008 collocated with ACM SIGMOD. Six instances of the workshop were successfully run until 2013. Five years later, in 2018, we revived the workshop in a new, biannual format. Today, the DBTest workshop consistently has high-quality submissions, expert presenters, and active participants across both academia and industry. Going forward, we plan to open the workshop up to an even more diverse audience, especially the research communities that focus on software testing and debugging in general, and not only on database systems.

Fluorescence dye adsorption assay to quantify carboxyl groups on the surface of poly(methyl methacrylate) microbeads.

Microbead-based assays have evolved into powerful tools for the multiplex detection of biomolecules. Analytes are captured by DNA or protein capture molecules which are coupled on microbead surfaces. A homogeneous carboxylation of microbeads is essential for the optimal and reproducible coupling of capture molecules and thus a prerequisite for an optimal multiplex microbead-based assay performance. We developed a simple fluorescence dye adsorption assay for the description of microbead carboxylation and for the prediction of coupling successes of capture molecules. Using the fluorescence dye SYTO-62 it is possible to quantify the degree of carboxylation of poly(methyl methacrylate) (PMMA) microbeads within 1 h in a multiplex format by fluorescence microscopy or flow cytometry. Compared to conventional bulk assays which only provide an average degree of carboxylation the main advantage of the SYTO-62 assay is the single microbead analysis and therefore the description of the qualitative distribution of carboxylation in microbead populations. The SYTO-62 assay is sensitive enough to even determine weak carboxylation. Also, the quality of microbeads can be evaluated. To our knowledge this is the first report which applies a reversible noncovalent fluorescent dye adsorption assay to quantify the degree of carboxylation on surfaces.

Analysis of three-dimensional biofilms on different material surfaces.

Biofilms cause complications and high costs in both industry and medicine. Of particular interest are bacterial infections of prosthetic materials, which usually cannot be eliminated due to the high antibiotic resistance known for bacteria forming biofilms. The search for new materials and coatings with lower colonization potential and antibacterial activity is of great importance to reduce biofilm formation. However, there is no standardized procedure to examine the colonization characteristics of bacteria in the biofilm state in situ. Here, we describe an automated epifluorescence microscopy system for the semi-quantitative analysis of three-dimensional (3D) biofilms on various surfaces. To analyze adherent bacteria, three materials (glass, steel and titanium) were incubated with bacteria in a flow chamber system. After fluorescence staining of the bacteria, automated image capturing, quantification of the bacteria, measurement of the colonized area and determination of the 3D biofilm height were carried out by using novel software. Furthermore, the materials were examined for their surface topography using white light scanning interferometry. Titanium compared to glass showed a significantly higher number of adherent bacteria. We argue that this was due to the higher microroughness of titanium. The colonized area was in accordance with the number of adherent bacteria and was also significantly larger on titanium coupons compared to glass. Maximum 3D biofilm height on glass coupons was significantly lower compared to the ones on steel and titanium. This novel method enables the standardized, automated investigation of the colonization with bacteria on different materials. This approach can considerably support the characterization of new material surfaces and their innovative coatings by analyzing the amount of attached bacteria and thickness of biofilms in situ and eliminates the need of conventional cultivation.

Association of biofilm formation and cytotoxic potential with multidrug resistance in clinical isolates of Pseudomonas aeruginosa.

Multidrug resistant (MDR) Pseudomonas aeruginosa having strong biofilm potential and virulence factors are a serious threat for hospitalized patients having compromised immunity. In this study, 34 P. aeruginosa isolates of human origin (17 MDR and 17 non-MDR clinical isolates) were checked for biofilm formation potential in enriched and minimal media. The biofilms were detected using crystal violet method and a modified software package of the automated VideoScan screening method. Cytotoxic potential of the isolates was also investigated on HepG2, LoVo and T24 cell lines using automated VideoScan technology. Pulse field gel electrophoresis revealed 10 PFGE types in MDR and 8 in non-MDR isolates. Although all isolates showed biofilm formation potential, strong biofilm formation was found more in enriched media than in minimal media. Eight MDR isolates showed strong biofilm potential in both enriched and minimal media by both detection methods. Strong direct correlation between crystal violet and VideoScan methods was observed in identifying strong biofilm forming isolates. High cytotoxic effect was observed by 4 isolates in all cell lines used while 6 other isolates showed high cytotoxic effect on T24 cell line only. Strong association of multidrug resistance was found with biofilm formation as strong biofilms were observed significantly higher in MDR isolates (p-value < 0.05) than non-MDR isolates. No significant association of cytotoxic potential with multidrug resistance or biofilm formation was found (p-value > 0.05). The MDR isolates showing significant cytotoxic effects and strong biofilm formation impose a serious threat for hospitalized patients with weak immune system.

Hypothesis on monochromatic vision in scorpionflies questioned by new transcriptomic data.

In the scorpionfly Panorpa, a recent study suggested monochromatic vision due to evidence of only a single opsin found in transcriptome data. To reconsider this hypothesis, the present study investigates opsin expression using transcriptome data of 21 species including representatives of all major lineages of scorpionflies (Mecoptera) and of three families of their closest relatives, the fleas (Siphonaptera). In most mecopteran species investigated, transcripts encode two opsins with predicted peak absorbances in the green, two in the blue, and one in the ultraviolet spectral region. Only in groups with reduced or absent ocelli, like Caurinus and Apteropanorpa, less than four visual opsin messenger RNAs have been identified. In addition, we found a Rh7-like opsin in transcriptome data derived from larvae of the mecopteran Nannochorista, and in two flea species. Peropsin expression was observed in two mecopterans. In light of these new data, we question the hypothesis on monochromatic vision in the genus Panorpa. In a broader phylogenetic perspective, it is suggested that the common ancestor of the monophyletic taxon Antliophora (Diptera, Mecoptera and Siphonaptera) possessed the full set of visual opsins, a Rh7-like opsin, and in addition a pteropsin as well as a peropsin. In the course of evolution individual opsins were likely lost in several lineages of this clade.

Covalent Attachment of Enzymes to Paper Fibers for Paper-Based Analytical Devices.

Due to its unique material properties, paper offers many practical advantages as a viable platform for sensing devices. In view of paper-based microfluidic biosensing applications, the covalent immobilization of enzymes with preserved functional activity is highly desirable and ultimately challenging. In the present manuscript, we report an efficient approach to achieving the covalent attachment of certain enzymes on paper fibers via a surface-bound network of hydrophilic polymers bearing protein-modifiable sites. This tailor-made macromolecular system consisting of polar, highly swellable copolymers is anchored to the paper exterior upon light-induced crosslinking of engineered benzophenone motifs. On the other hand, this framework contains active esters that can be efficiently modified by the nucleophiles of biomolecules. This strategy allowed the covalent immobilization of glucose oxidase and horseradish peroxidase onto cotton linters without sacrificing their bioactivities and performance upon surface binding. As a proof-of-concept application, a microfluidic chromatic paper-based glucose sensor was developed and achieved successful glucose detection in a simple yet efficient cascade reaction.

Adhesion of Salmonella to Pancreatic Secretory Granule Membrane Major Glycoprotein GP2 of Human and Porcine Origin Depends on FimH Sequence Variation.

Bacterial host tropism is a primary determinant of the range of host organisms they can infect. Salmonella serotypes are differentiated into host-restricted and host-adapted specialists, and host-unrestricted generalists. In order to elucidate the underlying molecular mechanisms of host specificity in Salmonella infection, we investigated the role of the intestinal host cell receptor zymogen granule membrane glycoprotein 2 (GP2), which is recognized by FimH adhesin of type 1 fimbriae found in Enterobacteriaceae. We compared four human and two porcine GP2 isoforms. Isoforms were expressed in Sf9 cells as well as in one human (HEp-2) and one porcine (IPEC-J2) cell line. FimH genes of 128 Salmonella isolates were sequenced and the 10 identified FimH variants were compared regarding adhesion (static adhesion assay) and infection (cell line assay) using an isogenic model. We expressed and characterized two functional porcine GP2 isoforms differing in their amino acid sequence to human isoforms by approximately 25%. By comparing all isoforms in the static adhesion assay, FimH variants were assigned to high, low or no-binding phenotypes. This FimH variant-dependent binding was neither specific for one GP2 isoform nor for GP2 in general. However, cell line infection assays revealed fundamental differences: using HEp-2 cells, infection was also FimH variant-specific but mainly independent of human GP2. In contrast, this FimH variant dependency was not obvious using IPEC-J2 cells. Here, we propose an alternative GP2 adhesion/infection mechanism whereby porcine GP2 is not a receptor that determined host-specificity of Salmonella. Salmonella specialists as well as generalists demonstrated similar binding to GP2. Future studies should focus on spatial distribution of GP2 isoforms in the human and porcine intestine, especially comparing health and disease.