Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea

Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties

The Impact of Metal Ion Exposure on the Cellular Behavior of Human Osteoblasts and PBMCs: In Vitro Analyses of Osteolytic Processes

Osteolysis in the periprosthetic tissue can be caused by metallic wear particles and ions that can originate from implant surface corrosion. These products influence cellular behavior and stimulate the expression of proinflammatory cytokines. The purpose of this study was to evaluate the impact of CoCr29Mo6 ions on cell survival, differentiation, and cytokine expression in human osteoblasts and peripheral blood mononuclear cells (PBMCs). Thus, we exposed cells with a mixture of 200 mu g/L ion solution and determined cell viability and apoptosis/necrosis. Gene expression analyses of osteoblastic and osteoclastic differentiation markers as well as pro-osteolytic mediators (IL-6, IL-8, TNF-alpha, MCP-1, MMP1, TIMP1) were performed. These markers were also investigated in mixed cultures of adherent and non-adherent PBMCs as well as in co-cultures of human osteoblasts and PBMCs. The ion solution induced necrosis in osteoblasts and PBMCs in single cultures. All examined mediators were highly expressed in the co-culture of osteoblasts and PBMCs whereas in the single cell cultures only IL-6, IL-8, and MMP1 were found to be stimulated. While the applied concentration of the CoCr29Mo6 ion solutions had only marginal effects on human osteoblasts and PBMCs alone, the co-culture may provide a comprehensive model to study osteolytic processes in response to Co and Cr ions

Severe polymicrobial and fungal periprosthetic osteomyelitis persisting after hip disarticulations treated with caspofungin in risk patients: a case series

Background!#!Periprosthetic fungal infections are considered rare and opportunistic infections. Treatment is difficult, and established standards do not yet exist. The choice of the appropriate antifungal drug might affect the patient outcome.!##!Cases!#!All the three cases presented showed polybacterial recurrent infection of the revision hip arthroplasty. All patients were of younger age, had multiple revisions of the endoprosthesis, each had a large partial femoral replacement greater than 40% of the femoral length, gentamycin-loaded cement, and a long anchoring distance of the used intramedullary stem. Due to the severe life-threatening infection with deep osteomyelitis, an amputation had to be performed. However, despite surgical intervention, the fungal dominated infection persisted. Finally, only the use of caspofungin allowed permanent infection control.!##!Conclusion!#!The polybacterial infection is driven by the symbiosis between fungi and bacteria. Therefore, eradication of the fungus is required to achieve elimination of the bacteria. Antimycotics of the echinocandin-class, such as caspofungin, may be considered as initial treatment

Net Promoter Score: a prospective, single-centre observational study assessing if a single question determined treatment success after primary or revision hip arthroplasty

Abstract Background Our study aimed to identify the relationship between treatment outcome assessed by patient-reported outcomes (PROMs) and satisfaction measured by calculation of the Net Promoter Score (NPS), which identifies promoters, following total hip arthroplasty (THA). The aim was to evaluate this association separately in primary and revision THA and to determine thresholds based on PROMs that identify detractors of the surgical procedure or the centre. Methods A total of 1,243 patients who underwent primary or revision THA at our hospital were asked to complete questionnaires of the Oxford Hip Score (OHS), Euroquol-5D (EQ-5D) and information on pain intensity preoperatively, three and 12 months after surgery. Postoperatively, the patients were additionally asked about their satisfaction with the procedure and the hospital by using three different NPS questions. The association between PROMs and NPS was evaluated based on group comparisons of primary or revision THA and receiver operating characteristics analysis (ROC) to determine threshold values. Results At 12 months the NPS of all three questions were invariably linked to treatment outcome in patients after primary THA and patients with a single revision. In these two treatment groups, promoters always showed significantly better PROM scores than detractors. The NPS score was always higher in the primary group in comparison to the single revision group, e.g. 66.4% would undergo the procedure again in the first group, while only 33.0% would opt for this in the latter group. The high thresholds for the PROMs at 12 months, that were calculated by ROC analysis to identify promoter/detractors, indicate that patients` satisfaction required very good joint function and pain relief. However, the NPS was not a suitable tool to identify patients who need further care in an early phase after surgery. Conclusions With NPS already a single question or a single parameter provides the desired information regarding patient satisfaction and also treatment success. Trial registration The study was approved by the Ethics Committee at the Medical Faculty of the University Rostock: “Ethikkommission an der Medizinischen Fakultät der Universität Rostock”, Address: St.-Georg Str. 108 18055 Rostock, Germany, reference number: A2015–0055

Potential probiotic Kluyveromyces marxianus B0399 modulates the immune response in Caco-2 cells and peripheral blood mononuclear cells and impacts the human gut microbiota in an in vitro colonic model system

Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 10(7) CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic

Inflammatory Response of Human Peripheral Blood Mononuclear Cells and Osteoblasts Incubated With Metallic and Ceramic Submicron Particles

Inflammatory reactions associated with osteolysis and aseptic loosening are the result of wear particles generated at the articulating surfaces of implant components. The aim of the present study was to analyze the biological response of human osteoblasts and peripheral blood mononuclear cells (PBMCs) after exposure to metallic and alumina ceramic particles regarding cellular differentiation, cytokine release, and monocyte migration. Cells were exposed to particles (0.01 and 0.05 mg/ml) from an alumina matrix composite (AMC) ceramic and a CoCr28Mo6 alloy with an average size of 0.5 µm over 48 and 96 h. The expression rates of osteogenic (Col1A1, ALP) and pro-osteoclastic (RANK, Trap5b) differentiation markers as well as pro-osteolytic mediators (MMP-1, TIMP-1, IL-6, IL-8, MCP-1) were determined and soluble protein concentrations of active MMP-1, IL-6, IL-8, and pro-collagen type 1 in cell culture supernatants were evaluated. Additionally, the capacity of particle-treated osteoblasts to attract potentially pro-inflammatory cells to the site of particle exposure was investigated by migration assays using osteoblast-conditioned media. The cellular morphology and metabolism of human osteoblasts and adherent PBMCs were influenced by particle type and concentration. In human osteoblasts, Col1A1 expression rates and protein production were significantly reduced after exposing cells to the lower concentration of cobalt-chromium (CoCr) and AMC particles. Exposure to AMC particles (0.01 mg/ml) resulted in increased mRNA levels of RANK and Trap5b in adherent PBMCs. For MMP-1 gene expression, elevated levels were more prominent after incubation with CoCr compared to AMC particles in osteoblasts, which was not reflected by the protein data. Interleukin (IL)-6 and IL-8 mRNA and protein were induced in both cell types after treatment with AMC particles, whereas exposure to CoCr particles resulted in significantly upregulated IL-6 and IL-8 protein contents in PBMCs only. Exposure of osteoblasts to CoCr particles reduced the chemoattractant potential of osteoblast-conditioned medium. Our results demonstrate distinct effects of AMC and CoCr particles in human osteoblasts and PBMCs. Complex cell and animal models are required to further evaluate the impact of cellular interactions between different cell types during particle exposure

Intraoperative damage to surgical gloves during various operations on the musculoskeletal system: a multicenter study

Introduction!#!Various orthopedic surgical procedures cause mechanical stress for gloves. In some cases, sharp-edged objects impact on the glove surfaces. The systematic description of lesions is still missing.!##!Methods!#!2289 gloves from 409 surgeries [primary hip and knee arthroplasties (PA), revisions arthroplasties (RA) and arthroscopic shoulder, hip and knee surgery (AY)] from 3 clinics were examined for lesions using water tightening test according to the European norm EN 455-1.!##!Results!#!Arthroscopies showed the lowest rate of operations with damaged gloves (6.9%). Depending on clinic, 32.7% and 59.2% of PA surgeries generated damaged gloves, while in RA, these numbers rose to 76.0% and 72.8%, respectively. In PA and RA, the most affected finger was the index finger, whereas in arthroscopies, more damage occurred on the middle finger and the thumb. The size of the lesions was rather small with the vast majority being 1 mm or 2 mm in size.!##!Conclusion!#!All investigated interventions led to glove lesions. With increasing mechanical stress, the number of glove defects increased. EN 455 does not account for the intraoperative tear risk. Stricter requirements for gloves should be introduced. Glove change intervals should be defined and implemented, and new materials should be developed

Influence of Conditioned Media on the Re-Differentiation Capacity of Human Chondrocytes in 3D Spheroid Cultures

A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO2 and 21% O2 in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation

Prospective data collection and analysis of perforations and tears of latex surgical gloves during primary endoprosthetic surgeries

Introduction: Surgical gloves are used to prevent contamination of the patient and the hospital staff with pathogens. The aim of this study was to examine the actual effectiveness of gloves by examining the damage (perforations, tears) to latex gloves during surgery in the case of primary hip and knee prosthesis implantation. Materials and methods: Latex surgical gloves used by surgeons for primary hip and knee replacement surgeries were collected directly after the surgery and tested using the watertightness test according to ISO EN 455-1:2000.Results: 540 gloves were collected from 104 surgeries. In 32.7% of surgeries at least one glove was damaged. Of all the gloves collected, 10.9% were damaged, mainly on the index finger. The size of the perforations ranged from ≤1 mm to over 5 mm. The surgeon’s glove size was the only factor that significantly influenced the occurrence of glove damage. Surgeon training level, procedure duration, and the use of bone cement had no significant influence.Conclusions: Our results highlight the high failure rate of surgical gloves. This has acute implications for glove production, surgical practice, and hygiene guidelines. Further studies are needed to detect the surgical steps, surface structures, and instruments that pose an increased risk for glove damage

Increased Diagnostic Certainty of Periprosthetic Joint Infections by Combining Microbiological Results with Histopathological Samples Gained via a Minimally Invasive Punching Technique

Background: The diagnosis of low-grade infections of endoprostheses is challenging. There are still no unified guidelines for standardised diagnostic approaches, recommendations are categorised into major and minor criteria. Additional histopathological samples might sustain the diagnosis. However, ambulatory preoperative biopsy collection is not widespread. Method: 102 patients with hip or knee endoprosthesis and suspected periprosthetic joint infection (PJI) were examined by arthrocentesis with microbiological sample and histopathological punch biopsy. The data were retrospectively analysed for diagnosis concordance. Results: Preoperative microbiology compared to intraoperative results was positive in 51.9% (sensitivity 51.9%, specificity 97.3%). In comparison of preoperative biopsy to intraoperative diagnostic results 51.9% cases were positive (sensitivity 51.9%, specificity 100.0%). The combination of preoperative biopsy and microbiology in comparison to intraoperative results was positive in 70.4% of the cases (sensitivity 70.4%, specificity 97.3%). Conclusion: The diagnosis of PJI is complex. One single method to reliably detect an infection is currently not available. With the present method histopathological samples might be obtained quickly, easily and safely for the preoperative detection of PJI. A combination of microbiological and histopathological sampling increases the sensitivity up to 18.5% to detect periprosthetic infection