Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440

<p>Abstract</p> <p>Background</p> <p>Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen <it>Pseudomonas aeruginosa </it>during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular <it>P. aeruginosa</it>. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly.</p> <p>Results</p> <p>Here, the metabolic engineering of a rhamnolipid producing <it>Pseudomonas putida </it>KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, <it>P. putida </it>KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria <it>Bacillus subtilis, Corynebacterium glutamicum</it>, and <it>Escherichia coli. P. putida </it>KT2440 expressing the <it>rhlAB</it>-genes from <it>P. aeruginosa </it>PAO1 produces mono-rhamnolipids of <it>P. aeruginosa </it>PAO1 type (mainly C₁₀:C₁₀). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of <it>P. aeruginosa </it>PAO1 at yields of about 0.15 g/g_glucosecorresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation.</p> <p>Conclusions</p> <p>A functional alternative to the pathogenic rhamnolipid producer <it>P. aeruginosa </it>was constructed and characterized. <it>P. putida </it>KT24C1 pVLT31_<it>rhlAB </it>featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer <it>P. aeruginosa</it>.</p

Equine Suture Exostosis: A Review of Cases from a Multicenter Retrospective Study

Simple summary Suture exostosis is a condition affecting the horse's head. The connections between the bone plates that form the horse's face have been shown to react to insult. Horses will then develop a swelling along the face that may be painful. Little is known about this condition and the present research project aimed to investigate horses presented to equine clinics with symptoms of the disorder. It was revealed that the condition can form following trauma, underlying sinus disease, following a surgery or without any apparent cause. Various treatment options to resolve the condition have been reported and the outcomes of those are described in the paper. Most consistently the proper diagnosis and identification and removal of potential bone sequestra are crucial for a timely resolution. Suture exostosis is an intriguing and not uncommon pathology that has to be included in the differential diagnosis for horses with swelling of the head. Although several singular case reports have been published, no large case series is available. The aim of this study is to report a multicentric retrospective collection of suture exostosis cases. Data concerning horses with suture exostosis in the facial region were collected retrospectively. Information regarding breed, age, gender, history, imaging findings, initiated treatment, response to treatment and follow up was recorded. One hundred and five cases of various breeds were reported. Analysis revealed the cases could be grouped into four entities: 45 developed following sino-nasal surgery, 23 following trauma, seven with underlying sinus pathology and 25 idiopathic. Treatment consisted of sequestra removal, plate fixation, antimicrobial and anti-inflammatory drugs or no treatment. Whereas initial localized pain fades within few days or weeks, resolution or reduction of the swelling was obtained in most cases after 3 months to 1.5 years. The etiopathogenesis of suture exostosis seems to consist of different entities. Identification of an underlying cause, particularly the presence of a bone sequester and infection is important to speed up resolution and before concluding an idiopathic case. When performing sinusotomies, it is important to provide as little trauma as possible to the surgical site in order to prevent suture exostosis as a complication

DKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker

The prominent role of the S100A8/S100A9-CD147 axis in the progression of penile cancer

Currently, no established biomarkers are recommended for the routine diagnosis of penile carcinoma (PeCa). The rising incidence of this human papillomavirus (HPV)–related cancer entity highlights the need for promising candidates. The Calprotectin subunits S100A8 and S100A9 mark myeloid-derived suppressor cells in other HPV-related entities while their receptor CD147 was discussed to identify patients with PeCa at a higher risk for poor prognoses and treatment failure. We thus examined their expression using immunohistochemistry staining of PeCa specimens from 74 patients on tissue microarrays of the tumor center, the invasion front, and lymph node metastases. Notably, whereas the tumor center was significantly more intensively stained than the invasion front, lymph node metastases were thoroughly positive for both S100 subunits. An HPV-positive status combined with an S100A8+S100A9+ profile was related with an elevated risk for metastases. We observed several PeCa specimens with S100A8+S100A9+-infiltrating immune cells overlapping with CD15 marking neutrophils. The S100A8+S100A9+CD15+ profile was associated with dedifferentiated and metastasizing PeCa, predominantly of HPV-associated subtype. These data suggest a contribution of neutrophil-derived suppressor cells to the progression of HPV-driven penile carcinogenesis. CD147 was elevated, expressed in PeCa specimens, prominently at the tumor center and in HPV-positive PeCa cell lines. CD147+HPV+ PeCa specimens were with the higher-frequency metastasizing cancers. Moreover, an elevated expression of CD147 of HPV-positive PeCa cell lines correlated negatively with the susceptibility to IgA-based neutrophil-mediated tumor cell killing. Finally, stratifying patients regarding their HPV/S100A8/S100A9/CD15/CD147 profile may help identify patients with progressing cancer and tailor immunotherapeutic treatment strategies

Serological immune response to cancer testis antigens in patients with pancreatic cancer

Serological screening approaches have allowed for the identification of a large number of potentially relevant tumor antigens in cancer patients. Within this group, cancer testis antigens represent promising targets for cancer immunotherapy, since they are widely expressed in a variety of human cancer entities. In pancreatic cancer, however, there are only few data available about the expression pattern and serological response to cancer testis antigens and other serological-defined tumor antigens. Therefore, we investigated the IgG antibody response against 11 cancer testis antigens (SCP-1, GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5) recombinantly expressed on yeast surface (RAYS) in patients with pancreatic cancer (n = 96), chronic pancreatitis (n = 18) and healthy donors (n = 48). We found in 14% of all patients antibody responses to SCP-1, but not to other cancer testis antigens (GAGE, LAGE-1a,-1b, CT-7, NY-ESO-1, SSX-1-5). Antibody response correlated with the expression of SCP-1 in the primary tumor of the respective patient as shown by RT-PCR, immunohistochemistry and Western blot. In contrast, no serological response to cancer testis antigens was observed in healthy donors. The humoral immune response against SCP-1 was associated with the size of tumor, but not with other clinico-pathological parameters such as histology, stage, presence of lymph node metastases, grading, age, gender or gemcitabine treatment. In conclusion, antibody response to cancer testis antigen SCP-1 is found in a proportion of pancreatic carcinoma patients. These results indicate that identification of additional tumor antigens by serological screening of tumor cDNA expression libraries by RAYS is a promising goal in pancreatic cancer

Körper von Gewicht. Zur Geschlechterdifferenz in den Ernährungs- und Körpernormen

Schiek D, Ullrich CG. Körper von Gewicht. Zur Geschlechterdifferenz in den Ernährungs- und Körpernormen. In: Zwick MM, Deuschle J, Renn O, eds. Übergewicht und Adipositas bei Kindern und Jugendlichen. Wiesbaden: VS Verlag für Sozialwissenschaften; 2011: 203-218

Suboptimal dosing of rituximab in male and female patients with DLBCL

To determine the effect of gender on outcome, the male hazard ratio for progression-free survival (HRPFS-male) was determined in patients with diffuse large B-cell lymphoma (DLBCL). In young patients (MapThera International Trial study), HRPFS-male was 1.3 (P = .092) without and 1.1 (P = .660) with rituximab. In elderly patients (RICOVER-60 study), HRPFS-male was 1.1 (P = .348) with CHOP but increased to 1.6 (P = .004) with R-CHOP. The similar improvements of outcome in young patients were associated with similar rituximab clearances in young males and females (9.89 vs 10.38 mL/h; P = .238), whereas the greater benefit for elderly females was associated with a slower rituximab clearance (8.47 vs 10.59 mL/h; P = .005) and hence higher serum levels and longer exposure times, attributable to an age-dependent (P = .004) decrease of rituximab clearance in females but not males. Compared with elderly females, all other subgroups had significantly faster rituximab clearances and hence appear to be suboptimally dosed when rituximab is given at 375 mg/m(2). Although early results of pharmacokinetic-based prospective trials designed to exploit the full therapeutic potential of rituximab suggest that increased doses and/or prolonged exposure times can improve the outcome of elderly males with DLBCL, further studies are warranted that address the optimization of rituximab dose and schedule in all subgroups of DLBCL patients

Optimization of Rituximab for the Treatment of Diffuse Large B-Cell Lymphoma (II): Extended Rituximab Exposure Time in the SMARTE-R-CHOP-14 Trial of the German High-Grade Non-Hodgkin Lymphoma Study Group

Purpose To study pharmacokinetics, toxicity, and efficacy of prolonged rituximab exposure in elderly patients with diffuse large B-cell lymphoma (DLBCL). Patients and Methods In the SMARTE-R-CHOP-14 trial, rituximab 375 mg/m(2) was administered, together with six cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone on a 14-day schedule (6xR-CHOP-14), on days -4, 0, 10, 29, 57, 99, 155, and 239. Pharmacokinetics and outcome were to be compared with those of patients who had received 6xR-CHOP-14 in combination with eight 2-week applications of rituximab in the RICOVER-60 (Rituximab With CHOP Over Age 60 Years) trial. Results The complete response (CR)/unconfirmed CR rate was 85% in 189 evaluable patients, 90% for 90 good-prognosis patients (International Prognostic Index [IPI], 1 or 2), and 81% for 99 poor-prognosis patients (IPI, 3 to 5); 3-year event-free survival (EFS) was 71%, 75%, and 67%, respectively; and 3-year overall survival (OS) was 84%, 88%, and 80%, respectively, with no differences between men and women. The preplanned historical comparison with 306 RICOVER-60 patients (good prognosis, n = 183; poor prognosis, n = 123) revealed no outcome differences for all and good-prognosis patients; however, the longer exposure time in SMARTE-R-CHOP-14 compared with RICOVER-60 was associated with better 3-year EFS (67% v 54%) and OS (80% v 67%) in poor-prognosis patients. Conclusion Extended rituximab exposure compared with eight 2-week applications in combination with 6xR-CHOP-14 significantly improved outcome of elderly poor-prognosis patients without increasing toxicity. To our knowledge, results obtained with the SMARTE-R-CHOP-14 rituximab schedule are the best reported for elderly patients with DLBCL to date. In the subgroup of poor-prognosis patients treated with extended rituximab exposure, the outcome seemed superior to that of a similar historical cohort of patients treated with 6xR-CHOP-14 plus 2-week rituximab, with similar toxicity. A randomized comparison of the two schedules is warranted. (C) 2014 by American Society of Clinical Oncolog