Plant invasion and fragmentation indirectly and contrastingly affect native plants and grassland arthropods.

Plant invasion and habitat fragmentation have a detrimental effect on biodiversity in nearly all types of ecosystems. We compared the direct and indirect effects of the invasion of the common milkweed (Asclepias syriaca) on biodiversity patterns in different-sized Hungarian forest-steppe fragments. We assessed vegetation structure, measured temperature and soil moisture, and studied organisms with different ecological roles in invaded and non-invaded sites of fragments: plants, bees, butterflies, flower-visiting wasps, flies, true bugs, and spiders. Temperature and soil moisture were lower in invaded than in non-invaded area. Milkweed had a positive effect on plant species richness and flower abundance. In contrast, we mainly found indirect effects of invasion on arthropods through alteration of physical habitat characteristics and food resources. Pollinators were positively affected by native flowers, thus, milkweed indirectly supported pollinators. Similarly, we found higher species richness of herbivores in invaded sites than control sites, as species richness of true bugs also increased with increasing plant species richness. Predators were positively affected by complex vegetation structure, higher soil moisture and lower temperature. Furthermore, increasing fragment size had a strong negative effect on spider species richness of non-invaded sites, but no effect in invaded sites. Especially, grassland specialist spiders were more sensitive to fragment size than generalists, whereas generalist spider species rather profited from invasion. Although milkweed invades natural areas, we did not identify strong negative effects of its presence on the diversity of the grassland biota. However, the supportive effect of milkweed on a few generalist species homogenises the communities. The rate of invasion might increase with increasing fragmentation, therefore we recommend eliminating invasive plants from small habitat fragments to preserve the native biota. Focusing also on generalist species and revealing the indirect effects of invasions are essential for understanding the invasion mechanisms and would support restoration efforts.

Brenneria bubanii sp. nov., isolated from decaying plant tissues.

Gram-negative, motile, rod-shaped bacterial strains (designated 4F2T and Kf) were isolated from decaying tissues of various deciduous tree species. Phylogenetic analyses based on their 16S rRNA gene sequences showed that the novel isolates belong to the genus Brenneria and showed highest (98.3 %) sequence similarity to Brenneria goodwinii. Isolate 4F2T formed a separate branch on the phylogenetic tree based on concatenated sequences of four housekeeping genes or whole genome sequences, clearly separate from Brenneria goodwinii, suggesting that novel isolates should belong to a novel species. Orthologous average nucleotide identity scores and in silico DNA-DNA hybridization values between isolate 4F2T and type strains of other species in the genus Brenneria were less than 85 and 30 %, respectively, significantly lower than the species boundary cut-off values (95 and 70 %). A negative reaction for ß-galactosidase, the ability to use dextrin and maltose as carbon sources, and an inability to use lactose are the main phenotypic characteristics that can be used to differentiate the novel isolates from B. goodwinii. Based on phenotypic and genotypic characteristics, isolates 4F2T and Kf belong to a novel species of the genus Brenneria, for which the name Brenneria bubanii sp. nov. is proposed. The type strain is 4F2T (=NCAIM B 02661T=LMG 32183T).

First Report of Colletotrichum godetiae Causing Anthracnose and Twig Blight on Persian Walnut in Hungary.

Persian walnut (Juglans regia L.) fruit with preharvest anthracnose symptoms, necrotic fruit stalks, and twigs with necrotic buds, and peaks were collected in a Hungarian orchard next to Nágocs, in September 2018. Disease incidence was approximately 15% on a Hungarian bred walnut cultivar 'Milotai 10'. Similar symptoms were found on Persian walnut in other locations (eg. Milota, Érd, Sarród, and Kocs). Acervuli were observed on necrotic lesions on fruit, and twigs with pale orange conidial masses. Conidia were hyaline, unicellular, and fusiform. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 15.9 ± 1.7 × 4.5 ± 0.4 µm, length/width ratio 1:0.3 (n=100). The fungus was isolated from conidial masses on potato dextrose agar (PDA) medium amended with Chlorampenicol (25 mg/L). A total of 12 isolates were obtained as pure cultures by single-spore isolations and incubated at 23°C in dark for 10 days. The colonies were white to gray or grayish-orange on the upper side and with black spots on the reverse side. The isolates showed morphological characteristics of Colletotrichum acutatum in sensu lato (Jayawardena et al. 2016). Molecular analyses were conducted to identify the exact species. Internal transcribed spacer (ITS) region, actin (ACT), and calmodulin (CAL) partial genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R and CAL1/CAL2 primers (White at al. 1990, Carbone and Kohn 1999, O'Donnell et al. 2000). The sequences of ITS region (GenBank Accession Nos: MK367398-99, MK367401-02) showed 100% identity with C. godetiae sequence. Based on ACT gene (GenBank Accession Nos: MK415991-92, MK415994-95) were 100% identity with the deposited C. godetiae type strains from walnut. The obtained sequences of CAL gene (GenBank Accession Nos: MK415998-99, MK416001-02) were same and showed 100% with other C. godetiae sequences from other host plants. The fungus was identified as Colletotrichum godetiae Neerg. Pathogenicity tests were accomplished in the field and under laboratory conditions (25°C on thermostat) on 10 green 'Milotai 10' walnut fruit, and 10 walnut twigs each. Tests were conducted on living trees, collected fruit, and two-year-old twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp tissues, which were then wrapped with wet cotton and parafilm. Wounded tissues on 5 fruit and 5 two-year-old twigs were treated with non-colonized PDA plugs as noninoculated controls. After 14 d necrotic lesions 9 to 17 mm in diameter developed on fruit on living trees. Lengths of 12 to 17 mm and width of 7 to 12 mm necrosis was measured on phloem of walnut twigs, and almost two times larger in cambium. No necrosis developed around control wounds. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues, isolates were identical morphologically and by sequence analysis of ITS region, ACT, and CAL partial genes to the original isolates. Damm et al. (2012) described two C. godetiae strains associated with walnut, one isolated in Austria and another one of unknown origin. An epidemic event of walnut anthracnose caused by Colletotrichum species mainly C. godetiae was reported in France (Da Lio et al. 2018). The pathogen was isolated from nuts, buds, insects, and stems. To our knowledge, this is the first report of anthracnose of walnut fruit caused by C. godetiae in Hungary. Anthracnose caused by C. godetiae, and previously reported C. fioriniae (Varjas et al. 2019) is becoming an increasing preharvest problem on Persian walnut in Hungary.

Nectar- and stigma exudate-specific expression of an acidic chitinase could partially protect certain apple cultivars against fire blight disease.

MAIN CONCLUSION: Certain apple cultivars accumulate to high levels in their nectar and stigma exudate an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora. To prevent microbial infections, flower nectars and stigma exudates contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia-resistant apples are not available, certain cultivars are tolerant. It was reported that in flower infection assay, the 'Freedom' cultivar was Erwinia tolerant, while the 'Jonagold' cultivar was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed, we found that an acidic chitinase III protein (Machi3-1) selectively accumulates to very high levels in the nectar and the stigma exudate of the 'Freedom' cultivar. We show that three different Machi3-1 alleles exist in apple cultivars and that only the 5B-Machi3-1 allele expresses the Machi3-1 protein in the nectar and the stigma exudate. We demonstrate that the 5B-Machi3-1 allele was introgressed from the Malus floribunda 821 clone into different apple cultivars including the 'Freedom'. Our data suggest that MYB-binding site containing repeats of the 5B-Machi3-1 promoter is responsible for the strong nectar- and stigma exudate-specific expression. As we found that in vitro, the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 protein could partially protect 5B-Machi3-1 allele containing cultivars against Erwinia by inhibiting the multiplication and biofilm formation of the pathogen in the stigma exudate and in the nectar.

Conversion arthroplasty of the hip: mid-term results are good.

The historical mid-term results of conversion total hip arthroplasty (THA) are acceptable; however, the complication rates are high. In total, 39 patients (45 hips) from 2 institutions underwent conversion THA from 1993-2006 and were retrospectively evaluated. The mean age was 48.3 years, the mean follow-up time was 8.7 years, and the mean duration arthrodesis prior to conversion THA was 18.2 years. The outcomes included operative time, blood loss, leg-length discrepancy (LLD), thigh circumference, Harris Hip Score (HHS), complications, and radiographic evaluation. A total of 34 THAs were cemented, and 11 were uncemented. The mean operative time was 102 minutes, and the mean blood loss was 1019 ml. The mean HHS improved from 32.4 to 82.5 (p<0.01). The mean LLD decreased from 4.2 to 1.1 cm, while the thigh circumference increased by a mean of 1.6 cm. Complications included: a positive Trendelenburg gait (6), early haematoma that required surgical evacuation (5), dislocation (2), deep infection (1), and early aseptic loosening of the components (2). In conclusion, the functional results of the conversion THA using predominantly cemented components are good at mid-term follow-up; although the complication rates remain higher than a standard primary THA, aseptic loosening rates of the cemented components is low at mid-term follow-up.

Effects of laser treatment on the expression of cytosolic proteins in the synovium of patients with osteoarthritis.

BACKGROUND AND OBJECTIVE: Low level laser therapy (LLLT) has been developed for non-invasive treatment of joint diseases. We have previously shown that LLLT influenced synovial protein expression in rheumatoid arthritis (RA). The aim of this study was to assess the effects of laser irradiation on osteoarthritic (OA) synovial protein expression. STUDY DESIGN/MATERIALS AND METHODS: The synovial membrane samples removed from the knees of 6 OA patients were irradiated ex vivo using near infrared diode laser (807-811 nm; 25 J/cm(2) ). An untreated sample taken from the same patient served as control. Synovial protein separation and identification were performed by two-dimensional differential gel electrophoresis and mass spectrometry, respectively. RESULTS: Eleven proteins showing altered expression due to laser irradiation were identified. There were three patients whose tissue samples demonstrated a significant increase (P < 0.05) in mitochondrial heat shock 60 kD protein 1 variant 1. The expression of the other proteins (calpain small subunit 1, tubulin alpha-1C and beta 2, vimentin variant 3, annexin A1, annexin A5, cofilin 1, transgelin, and collagen type VI alpha 2 chain precursor) significantly decreased (P < 0.05) compared to the control samples. CONCLUSIONS: A single diode laser irradiation of the synovial samples of patients with osteoarthritis can statistically significantly alter the expression of some proteins in vitro. These findings provide some more evidence for biological efficacy of LLLT treatment, used for osteoarthritis.

The recently identified hexosaminidase D enzyme substantially contributes to the elevated hexosaminidase activity in rheumatoid arthritis.

Since the 1970s, numerous reports have described elevated hexosaminidase activities in rheumatoid arthritis. However, due to the overlapping substrate specificities of different hexosaminidases, identification of the exact enzyme(s) responsible for the elevated activity remains incomplete. In this work we tested if the recently described enzyme, hexosaminidase D was expressed in human arthritic joints, and could contribute to the elevated hexosaminidase activity in rheumatoid arthritis. Thermostable ß-d-N-acetyl-galactosaminidase (hexosaminidase D) activities were determined in synovial fluid samples, synovial membranes, synovial fibroblast cell strains and synovial fibroblast-derived extracellular vesicles of patients with rheumatoid arthritis and osteoarthritis using chromogenic substrates. Expression of the HEXDC gene was detected both in steady state and in TGF-ß treated synovial fibroblasts by real time PCR. Strikingly, hexosaminidase D accounted for approximately 50% of the total ß-N-acetyl-galactosaminidase activity in synovial membranes and synovial fibroblasts, and it was responsible for the vast majority of the ß-d-N-acetyl-galactosaminidase activity in synovial fluid samples. TGF-ß downregulated the expression of hexosaminidase D in synovial fibroblasts dose-dependently. Of note, significant activity of hexosaminidase D was also found in association with extracellular vesicles released by synovial fibroblasts. This first study that describes the expression and disease relevance of the HEXDC gene in humans demonstrates the expression of this novel enzyme within the joints, and suggests that its activity may significantly contribute to the overall local exoglycosidase activity.

Lonsdalea quercina subsp. populi subsp. nov., isolated from bark canker of poplar trees.

Seven Gram-negative bacterial strains were isolated from oozing bark canker of poplar (Populus × euramericana) trees in Hungary. They showed high (>98.3%) 16S rRNA gene sequence similarity to Lonsdalea quercina; however, they differed from this species in several phenotypic characteristics. Multilocus sequence analysis based on three housekeeping genes (gyrB, atpD and infB) revealed, and DNA-DNA hybridization analysis confirmed, that this group of bacterial strains forms a distinct lineage within the species Lonsdalea quercina. A detailed study of phenotypic and physiological characteristics confirmed the separation of isolates from poplars from other subspecies of L. quercina; therefore, a novel subspecies, Lonsdalea quercina subsp. populi, type strain NY060(T) (=DSM 25466(T)=NCAIM B 02483(T)), is proposed.

Ex vivo soft-laser treatment inhibits the synovial expression of vimentin and α-enolase, potential autoantigens in rheumatoid arthritis.

BACKGROUND: Soft-laser therapy has been used to treat rheumatic diseases for decades. The major effects of laser treatment may be dependent not on thermal mechanisms but rather on cellular, photochemical mechanisms. However, the exact cellular and molecular mechanisms of action have not been elucidated. OBJECTIVE: The aim of this study was to investigate the ex vivo effects of low-level laser treatment (with physical parameters similar to those applied previously) on protein expression in the synovial membrane in rheumatoid arthritis (RA). DESIGN: Synovial tissues were laser irradiated, and protein expression was analyzed. METHODS: Synovial membrane samples obtained from 5 people who had RA and were undergoing knee surgery were irradiated with a near-infrared diode laser at a dose of 25 J/cm(2) (a dose used in clinical practice). Untreated synovial membrane samples obtained from the same people served as controls. Synovial protein expression was assessed with 2-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry. RESULTS: The expression of 12 proteins after laser irradiation was different from that in untreated controls. Laser treatment resulted in the decreased expression of α-enolase in 2 samples and of vimentin and precursors of haptoglobin and complement component 3 in 4 samples. The expression of other proteins, including 70-kDa heat shock protein, 96-kDa heat shock protein, lumican, osteoglycin, and ferritin, increased after laser therapy. LIMITATIONS: The relatively small sample size was a limitation of the study. CONCLUSIONS: Laser irradiation (with physical parameters similar to those used previously) resulted in decreases in both α-enolase and vimentin expression in the synovial membrane in RA. Both proteins have been considered to be important autoantigens that are readily citrullinated and drive autoimmunity in RA. Other proteins that are expressed differently also may be implicated in the pathogenesis of RA. Our results raise the possibility that low-level laser treatment of joints affected with RA may be effective, at least in part, by suppressing the expression of autoantigens. Further studies are needed.

Gene expression and activity of cartilage degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts.

INTRODUCTION: Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints. METHODS: Expressions of beta-D-hexosaminidase, beta-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. beta-D-Glucuronidase, beta-D-glucosaminidase and beta-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity. RESULTS: According to our data, beta-D-hexosaminidase, beta-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the beta-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-beta1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFalpha, IL-1beta and IL-17. CONCLUSIONS: According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.

Chondrogenic potential of mesenchymal stem cells from patients with rheumatoid arthritis and osteoarthritis: measurements in a microculture system.

BACKGROUND: Mesenchymal stem cells (MSCs) have the potential to differentiate into distinct mesenchymal tissues; including cartilage and bone, they can be an attractive cell source for cartilage tissue engineering approaches. Our objective here was to compare the in vitro chondrogenic potential of MSCs isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) with cells from normal donors. METHODS: Marrow samples were removed during bone surgery and adherent cell cultures were established. The cells were then passed into a newly developed microaggregate culture system in a medium containing transforming growth factor beta3, insulin, dexamethasone and/or demineralized bone matrix. In vitro chondrogenic activity was measured as metabolic sulfate incorporation and type II collagen expression in pellet cultures. RESULTS: Culture-expanded MSCs from RA and OA patients did not differ significantly from the normal population with respect to their chondrogenic potential in vitro. Capability of total protein and proteoglycan synthesis as well as collagen II mRNA expression by cell aggregates was similar for all cell preparations in the presence of the appropriate growth and differentiation factors. Chondroprotective drugs such as chondroitin sulfate and glucosamine enhanced, whereas chloroquine inhibited chondrogenesis in normal donor-derived or patient-derived MSC cultures. Galectin-1, a beta-galactoside-binding protein with marked anti-inflammatory activity, stimulated the chondrogenic differentiation of mesenchymal cells in low (<2 microg/ml) concentration. DISCUSSION: These findings show that MSCs from RA and OA patients possess similar chondrogenic potential as MSCs isolated from healthy donors, therefore these cells may serve as a potential new prospect in cartilage replacement therapy.

Photorhabdus temperata subsp. cinerea subsp. nov., isolated from Heterorhabditis nematodes.

During the characterization of symbiotic bacteria of Hungarian entomopathogenic nematode isolates, a number of bacteria (including strain 3107(T)) isolated from Heterorhabditis downesi and Heterorhabditis megidis showed only moderate 16S rRNA gene sequence similarity to the type strains of all described Photorhabdus species and subspecies. On the basis of the 16S rRNA gene sequence, the phylogenetic relationships of these isolates were uncertain, because of the low bootstrap values. Using gyrB sequences for phylogenetic analysis, these isolates were shown to be part of the species Photorhabdus temperata, with clear separation from both Palaearctic and American strains (phylogenetic distances are 6.9 and 7.9 %, respectively). Physiological properties and carbon-source utilization profiles supported the phylogenetic position of these strains; therefore, a novel subspecies, Photorhabdus temperata subsp. cinerea subsp. nov. is proposed, with the type strain 3107(T) (=DSM 19724(T) =NCAIM B 02271(T)).

Electron microscopic characteristics of beta2-microglobulin amyloid deposits in long-term haemodialysis.

The electron microscopic features of beta2-microglobulin amyloid, deposited in the synovial membrane, are presented and discussed. The patient, a 69-year-old woman underwent chronic hemodialysis for 3 years. Because of constant pain and destructive arthropathy, endoprosthesis of the hip joints were implanted. Extra- and intracellular filamentous-fibrillar amyloid deposits have been demonstrated in ultrathin sections. The extracellular amyloid deposits showed a loose, filamentous or fibrillar structure at the periphery and a dense central core. The loose, filamentous structure may represent an early stage of fresh, newly deposited beta2-microglobulin amyloid, while the condensed and fragmented amyloid filaments may be an advanced "mature" stage of amyloid deposition.

Synovial fluid exoglycosidases are predictors of rheumatoid arthritis and are effective in cartilage glycosaminoglycan depletion.

OBJECTIVE: To analyze enzymes involved in joint damage by simultaneous investigation of glycosidases and matrix metalloproteinases (MMPs) in patients with various joint diseases. METHODS: Activities of glycosidases (beta-D-glucuronidase, beta-D-N-acetyl-glucosaminidase, beta-D-N-acetyl-galactosaminidase, beta-D-galactosidase, and alpha-D-mannosidase) were tested at an acidic pH as well as at the original pH of the synovial fluid (SF) samples in parallel with activities of MMP-1 and MMP-9. RESULTS: Patients with rheumatoid arthritis (RA) were characterized by significantly elevated activities of beta-D-glucuronidase and beta-D-N-acetyl-glucosaminidase in SF compared with patients with osteoarthritis, seronegative spondylarthritis, or acute sports injury. To select the best predictor for distinguishing among patient groups, a stepwise logistic regression analysis was performed; the strongest association was found to be between RA and beta-D-glucuronidase/beta-D-N-acetyl-glucosaminidase activities (measured at the pH of the SF). Further, a significant correlation was observed between the activity of SF beta-D-N-acetyl-glucosaminidase and the level of rheumatoid factor. In vitro digestion of human hyaline cartilage samples revealed that the dominant glycosidases, alone or in combination with MMPs, proved to be effective in depleting glycosaminoglycans (GAGs) from cartilage. CONCLUSION: These results suggest that exoglycosidases, which are present in the SF of RA patients, may contribute to the depletion of GAGs from cartilage and thereby facilitate the invasion of synovial cells and their attachment to cartilage in RA.