Primary osteoarthritis chondrocyte map of chromatin conformation reveals novel candidate effector genes.

OBJECTIVES: Osteoarthritis is a complex disease with a huge public health burden. Genome-wide association studies (GWAS) have identified hundreds of osteoarthritis-associated sequence variants, but the effector genes underpinning these signals remain largely elusive. Understanding chromosome organisation in three-dimensional (3D) space is essential for identifying long-range contacts between distant genomic features (e.g., between genes and regulatory elements), in a tissue-specific manner. Here, we generate the first whole genome chromosome conformation analysis (Hi-C) map of primary osteoarthritis chondrocytes and identify novel candidate effector genes for the disease. METHODS: Primary chondrocytes collected from 8 patients with knee osteoarthritis underwent Hi-C analysis to link chromosomal structure to genomic sequence. The identified loops were then combined with osteoarthritis GWAS results and epigenomic data from primary knee osteoarthritis chondrocytes to identify variants involved in gene regulation via enhancer-promoter interactions. RESULTS: We identified 345 genetic variants residing within chromatin loop anchors that are associated with 77 osteoarthritis GWAS signals. Ten of these variants reside directly in enhancer regions of 10 newly described active enhancer-promoter loops, identified with multiomics analysis of publicly available chromatin immunoprecipitation sequencing (ChIP-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) data from primary knee chondrocyte cells, pointing to two new candidate effector genes SPRY4 and PAPPA (pregnancy-associated plasma protein A) as well as further support for the gene SLC44A2 known to be involved in osteoarthritis. For example, PAPPA is directly associated with the turnover of insulin-like growth factor 1 (IGF-1) proteins, and IGF-1 is an important factor in the repair of damaged chondrocytes. CONCLUSIONS: We have constructed the first Hi-C map of primary human chondrocytes and have made it available as a resource for the scientific community. By integrating 3D genomics with large-scale genetic association and epigenetic data, we identify novel candidate effector genes for osteoarthritis, which enhance our understanding of disease and can serve as putative high-value novel drug targets.

The impact of insect egg deposition on Pinus sylvestris transcriptomic and phytohormonal responses to larval herbivory.

Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and-as expected-to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.

Priming of Arabidopsis resistance to herbivory by insect egg deposition depends on the plant's developmental stage.

While traits of plant resistance to herbivory often change during ontogeny, it is unknown whether the primability of this resistance depends on the plant's developmental stage. Resistance in non-flowering Arabidopsis thaliana against Pieris brassicae larvae is known to be primable by prior egg deposition on leaves. We investigated whether this priming effect is maintained in plants at the flowering stage. Larval performance assays revealed that flowering plants' resistance to herbivory was not primable by egg deposition. Accordingly, transcriptomes of flowering plants showed almost no response to eggs. In contrast, egg deposition on non-flowering plants enhanced the expression of genes induced by subsequent larval feeding. Strikingly, flowering plants showed constitutively high expression levels of these genes. Larvae performed generally worse on flowering than on non-flowering plants, indicating that flowering plants constitutively resist herbivory. Furthermore, we determined the seed weight in regrown plants that had been exposed to eggs and larvae during the non-flowering or flowering stage. Non-flowering plants benefitted from egg priming with a smaller loss in seed yield. The seed yield of flowering plants was unaffected by the treatments, indicating tolerance towards the larvae. Our results show that the primability of anti-herbivore defences in Arabidopsis depends on the plant's developmental stage.

Pine defense against eggs of an herbivorous sawfly is elicited by an annexin-like protein present in egg-associated secretion.

Known elicitors of plant defenses against eggs of herbivorous insects are low-molecular-weight organic compounds associated with the eggs. However, previous studies provided evidence that also proteinaceous compounds present in secretion associated with eggs of the herbivorous sawfly Diprion pini can elicit defensive responses in  Pinus sylvestris. Pine responses induced by the proteinaceous secretion are known to result in enhanced emission of (E)-ß-farnesene, which attracts egg parasitoids killing the eggs. Here, we aimed to identify the defense-eliciting protein and elucidate its function. After isolating the defense-eliciting protein from D. pini egg-associated secretion by ultrafiltration and gel electrophoresis, we identified it by MALDI-TOF mass spectrometry as an annexin-like protein, which we named 'diprionin'. Further GC-MS analyses showed that pine needles treated with heterologously expressed diprionin released enhanced quantities of (E)-ß-farnesene. Our bioassays confirmed attractiveness of diprionin-treated pine to egg parasitoids. Expression of several pine candidate genes involved in terpene biosynthesis and regulation of ROS homeostasis was similarly affected by diprionin and natural sawfly egg deposition. However, the two treatments had different effects on expression of pathogenesis-related genes (PR1, PR5). Diprionin is the first egg-associated proteinaceous elicitor of indirect plant defense against insect eggs described so far.

Priming by Timing: Arabidopsis thaliana Adjusts Its Priming Response to Lepidoptera Eggs to the Time of Larval Hatching.

Plants can respond to eggs laid by herbivorous insects on their leaves by preparing (priming) their defense against the hatching larvae. Egg-mediated priming of defense is known for several plant species, including Brassicaceae. However, it is unknown yet for how long the eggs need to remain on a plant until a primed defense state is reached, which is ecologically manifested by reduced performance of the hatching larvae. To address this question, we used Arabidopsis thaliana, which carried eggs of the butterfly Pieris brassicae for 1-6 days prior to exposure to larval feeding. Our results show that larvae gained less biomass the longer the eggs had previously been on the plant. The strongest priming effect was obtained when eggs had been on the plant for 5 or 6 days, i.e., for (almost) the entire development time of the Pieris embryo inside the egg until larval hatching. Transcript levels of priming-responsive genes, levels of jasmonic acid-isoleucine (JA-Ile), and of the egg-inducible phytoalexin camalexin increased with the egg exposure time. Larval performance studies on mutant plants revealed that camalexin is dispensable for anti-herbivore defense against P. brassicae larvae, whereas JA-Ile - in concert with egg-induced salicylic acid (SA) - seems to be important for signaling egg-mediated primed defense. Thus, A. thaliana adjusts the kinetics of its egg-primed response to the time point of larval hatching. Hence, the plant is optimally prepared just in time prior to larval hatching.

Defense of Scots pine against sawfly eggs (Diprion pini) is primed by exposure to sawfly sex pheromones.

Plants respond to insect infestation with defenses targeting insect eggs on their leaves and the feeding insects. Upon perceiving cues indicating imminent herbivory, such as damage-induced leaf odors emitted by neighboring plants, they are able to prime their defenses against feeding insects. Yet it remains unknown whether plants can amplify their defenses against insect eggs by responding to cues indicating imminent egg deposition. Here, we tested the hypothesis that a plant strengthens its defenses against insect eggs by responding to insect sex pheromones. Our study shows that preexposure of Pinus sylvestris to pine sawfly sex pheromones reduces the survival rate of subsequently laid sawfly eggs. Exposure to pheromones does not significantly affect the pine needle water content, but results in increased needle hydrogen peroxide concentrations and increased expression of defense-related pine genes such as SOD (superoxide dismutase), LOX (lipoxygenase), PAL (phenylalanine ammonia lyase), and PR-1 (pathogenesis related protein 1) after egg deposition. These results support our hypothesis that plant responses to sex pheromones emitted by an herbivorous insect can boost plant defensive responses to insect egg deposition, thus highlighting the ability of a plant to mobilize its defenses very early against an initial phase of insect attack, the egg deposition.

Early plant defence against insect attack: involvement of reactive oxygen species in plant responses to insect egg deposition.

MAIN CONCLUSION: Pinus sylvestris responds to insect egg deposition by ROS accumulation linked with reduced activity of the ROS scavenger catalase. Egg mortality in needles with hypersensitive response (HR)-like symptoms is enhanced. Aggressive reactive oxygen species (ROS) play an important role in plant defence against biotic stressors, including herbivorous insects. Plants may even generate ROS in response to insect eggs, thus effectively fighting against future larval herbivory. However, so far nothing is known on how ROS-mediated plant defence against insect eggs is enzymatically regulated. Neither do we know how insects cope with egg-induced plant ROS. We addressed these gaps of knowledge by studying the activities of ROS-related enzymes in Pinus sylvestris deposited with eggs of the herbivorous sawfly Diprion pini. This species cuts a slit into pine needles and inserts its eggs into the needle tissue. About a quarter of egg-deposited needles show chlorotic tissue at the oviposition sites, indicating hypersensitive response-like direct defence responses resulting in reduced larval hatching from eggs. Hydrogen peroxide and peroxidase sensitive staining of sections of egg-deposited pine needles revealed the presence of hydrogen peroxide and peroxidase activity in needle tissue close to the eggs. Activity of ROS-producing NADPH-oxidase did not increase after egg deposition. However, the activity of the ROS-detoxifying enzyme catalase decreased after egg deposition and ovipositional wounding of needles. These results show that local ROS accumulation at the oviposition site is not caused by increased NADPH-oxidase activity, but reduced activity of pine needle catalase may contribute to it. However, our data suggest that pine sawflies can counteract the egg deposition-induced hydrogen peroxide accumulation in pine needles by high catalase activity in their oviduct secretion which is released with the eggs into pine tissue.

Addressing the heterogeneous IT landscape of bioanalytical laboratories to increase efficiency of regulatory reporting and data analysis.

Modern bioanalytical laboratories are operating heterogeneous IT systems to satisfy process-related data capturing and processing. Whenever a cross-system perspective is desirable, for example, to answer 'quality-by-design'-related questions or populate submission documents, manual processes dominate these organizations. This article discusses the origin of this situation and the typical approach as well as a state-of-the-art approach to resolve cross-system obstacles.

A 'rare biosphere' microorganism contributes to sulfate reduction in a peatland.

Methane emission from peatlands contributes substantially to global warming but is significantly reduced by sulfate reduction, which is fuelled by globally increasing aerial sulfur pollution. However, the biology behind sulfate reduction in terrestrial ecosystems is not well understood and the key players for this process as well as their abundance remained unidentified. Comparative 16S rRNA gene stable isotope probing (SIP) in the presence and absence of sulfate indicated that a Desulfosporosinus species, which constitutes only 0.006% of the total microbial community 16S rRNA genes, is an important sulfate reducer in a long-term experimental peatland field site. Parallel SIP using dsrAB (encoding subunit A and B of the dissimilatory (bi)sulfite reductase) identified no additional sulfate reducers under the conditions tested. For the identified Desulfosporosinus species a high cell-specific sulfate reduction rate of up to 341 fmol SO4²â» cell⁻¹ day⁻¹ was estimated. Thus, the small Desulfosporosinus population has the potential to reduce sulfate in situ at a rate of 4.0-36.8 nmol (g soil w. wt.)⁻¹ day⁻¹, sufficient to account for a considerable part of sulfate reduction in the peat soil. Modeling of sulfate diffusion to such highly active cells identified no limitation in sulfate supply even at bulk concentrations as low as 10 µM. Collectively, these data show that the identified Desulfosporosinus species, despite being a member of the 'rare biosphere', contributes to an important biogeochemical process that diverts the carbon flow in peatlands from methane to CO2 and, thus, alters their contribution to global warming.