HinT proteins and their putative interaction partners in Mollicutes and Chlamydiaceae

BACKGROUND: HinT proteins are found in prokaryotes and eukaryotes and belong to the superfamily of HIT proteins, which are characterized by an histidine-triad sequence motif. While the eukaryotic variants hydrolyze AMP derivates and modulate transcription, the function of prokaryotic HinT proteins is less clearly defined. In Mycoplasma hominis, HinT is concomitantly expressed with the proteins P60 and P80, two domains of a surface exposed membrane complex, and in addition interacts with the P80 moiety. RESULTS: An cluster of hitABL genes, similar to that of M. hominis was found in M. pulmonis, M. mycoides subspecies mycoides SC, M. mobile and Mesoplasma florum. RT-PCR analyses provided evidence that the P80, P60 and HinT homologues of M. pulmonis were polycistronically organized, suggesting a genetic and physical interaction between the proteins encoded by these genes in these species. While the hit loci of M. pneumoniae and M. genitalium encoded, in addition to HinT, a protein with several transmembrane segments, the hit locus of Ureaplasma parvum encoded a pore-forming protein, UU270, a P60 homologue, UU271, HinT, UU272, and a membrane protein of unknown function, UU273. Although a full-length mRNA spanning the four genes was not detected, amplification of all intergenic regions from the center of UU270 to the end of UU273 by RT-PCR may be indicative of a common, but unstable mRNA. In Chlamydiaceae the hit gene is flanked upstream by a gene predicted to encode a metal dependent hydrolase and downstream by a gene putatively encoding a protein with ARM-repeats, which are known to be involved in protein-protein interactions. In RT-PCR analyses of C. pneumoniae, regions comprising only two genes, Cp265/Cp266 and Cp266/Cp267 were able to be amplified. In contrast to this in vivo interaction analysis using the yeast two-hybrid system and in vitro immune co-precipitation revealed an interaction between Cp267, which contains the ARM repeats, Cp265, the predicted hydrolase, and Cp266, the HinT protein. CONCLUSION: In the Mollicutes HinT proteins were shown to be linked with membrane proteins while in the Chlamydiaceae they were genetically and physically associated with cytoplasmic proteins, one of which is predicted to be a metal-dependent phosphoesterase. Future work will elucidate whether these differing associations indicate that HinT proteins have evolved independently or are indeed two hotspots of a common sphere of action of bacterial HinT proteins

Effekte biomechanischer Kräfte auf die Morphologie und glatt-muskuläre Differenzierung humaner mesenchymaler Stromazellen des Knochenmarks

In der vorliegenden Versuchsreihe wird der Einfluss von biomechanischer Stimulation auf humane Knochenmarksstromazellen (hbmMSC) in der glatt-muskulären Differenzierung untersucht. bmMSC von Patienten, die sich einer Hüft-Gelenksimplantation unterziehen (n=8) werden in einem Medium aus DMEM, gepooltem Plasma und Plättchenlysat expandiert und in den Passagen 2-5 mit einer Dichte von 5000 Zellen/cm2 auf kompaktierten Kollagen-I-Hydrogelen mit einer Konzentration von 80 mg/ml ausgesät. Das Hydrogel wird hierbei nach einer für die myogene Differenzierung optimalisierten Steifigkeit ausgewählt. Nach einer Adhärenzzeit von 4 Tagen wird die Membranen an einem bzw. zwei konsekutiven Tagen für jeweils eine Stunde mit 2,5% bzw. 5% Substratelongation mit dem ElectroForce 5210 uniaxial gestrecht. Wie auf den unstimulierten Kontrollen werden die Zellen bezüglich Zellzahl und Morphologie (Formfaktoren) mittels Image J quantifiziert. Außerdem werden Marker der glattmuskulären Differenzierung (ACTA-2, TGLN, CNN-1) per PCR und Immunfloureszenz untersucht. In der PCR zeigt sich unter 5%-Stretch ein signifikanter Anstieg der frühen glattmuskulären Marker α-Actin und Transgelin (p<0.05) an Tag 5 sowie ein transienter Anstieg des intermediären Markers Calponin I an Tag 4. Somit gelingt durch die Kombination von optimiertem outside-in (Steifigkeits-induziert) und inside-out (biomechanisch-induziertem) Stress die Induktion eines glatt-muskulären intermediären Markerprofils. Diese Ergebnisse sind besonders bemerkenswert, da mit dem kompaktierten Kollagen-I-Hydrogel ein pro-osteogenes, pro-fibrotisches und pro-synthetisches Substrat gewählt wurde. So unterstreicht die Versuchsreihe den elementaren Einfluss von mechanischem Stress und dokumentiert, dass bio-mechanische Stimulation das Verhalten von MSC „umprogrammieren“ kann. Somit könnte man in Zukunft durch Steuerung der MSC-Morphologie und den gezeigten Ansatz aus Kombination von outside-in und inside-out Stress, gezielt die myogene Differenzierung von MSC promoten, ohne andere Kofaktoren wie z.B. die Beschichtung oder Wachstumsfaktoren berücksichtigen zu müssen

The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffnessâ defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverseâ transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretchâ induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterialâ associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/1/term2263.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/2/term2263_am.pd

Desire for biological parenthood and patient counseling on the risk of infertility among adolescents and adults with hemoglobinopathies.

BACKGROUND Both diagnosis and treatment of hemoglobinopathies have been associated with an increased risk of fertility impairment. German guidelines recommend annual monitoring of fertility parameters to enable early detection of fertility impairment and/or to offer fertility preservation (FP) when indicated. We explored the general desire for parenthood, the frequency of recalling fertility counseling and testing, and the utilization of FP in adolescents and adults with hemoglobinopathies. PROCEDURE In a cross-sectional study, patients aged 12-50 years, treated in Germany, Austria, or Switzerland, were surveyed on fertility-related aspects. Medical data, including fertility testing results, were collected from patient records. RESULTS Overall, 116/121 eligible patients, diagnosed with sickle cell disease (70.7%), thalassemia (27.6%), or other hemoglobinopathy (1.7%), participated in our study (57.8% female, median age 17.0 years, range 12-50 years). All participants required treatment of the underlying hemoglobinopathy: 68.1% received hydroxyurea, 25.9% required regular blood transfusions, and 6.0% underwent hematopoietic stem cell transplantation (HSCT). Most patients (82/108, 75.9%) stated a considerable to strong desire for (future) parenthood, independent of sex, education, diagnosis, or subjective health status. Fertility counseling was only recalled by 32/111 patients (28.8%) and least frequently by younger patients (12-16 years) or those treated with regular blood transfusions or hydroxyurea. While fertility testing was documented for 59.5% (69/116) in medical records, only 11.6% (13/112) recalled previous assessments. FP was only used by 5.4% (6/111) of patients. CONCLUSION Most patients with hemoglobinopathies wish to have biological children, yet only few recalled fertility counseling and testing. Adequate patient counseling should be offered to all patients at risk for infertility

Ramucirumab plus docetaxel versus placebo plus docetaxel in patients with locally advanced or metastatic urothelial carcinoma after platinum-based therapy (RANGE): a randomised, double-blind, phase 3 trial

Few treatments with a distinct mechanism of action are available for patients with platinum-refractory advanced or metastatic urothelial carcinoma. We assessed the efficacy and safety of treatment with docetaxel plus either ramucirumab-a human IgG1 VEGFR-2 antagonist-or placebo in this patient population

Denosumab treatment in the management of patients with advanced prostate cancer: clinical evidence and experience

Osteoprotective therapies have become an essential component in the management of advanced prostate cancer (PC) patients as bone metastases (BMs) have a major impact on morbidity and mortality. Denosumab is a fully humanized antibody targeting the receptor activator of nuclear factor κB ligand (RANKL), which has been approved by the European Medicines Agency (EMA) in Europe and the United States (US) Food and Drug Administration (FDA) in the US for prevention of skeletal-related events (SREs) in patients with solid tumors and BMs, including PC. The clinical settings in which PC patients should be treated with denosumab are still discussed controversially. In a phase III study, denosumab significantly delayed SREs compared with zoledronic acid (ZA) in patients with metastatic castration-resistant PC (CRPC). In addition, denosumab showed superior effects on pain and health-related quality of life (QoL) in these patients. In patients with nonmetastatic CRPC, denosumab has been proven to significantly increase bone metastases-free survival. However, no significant benefits on cancer-specific and overall survival were observed and denosumab was not approved by the US FDA and EMA in this context. The effectiveness of denosumab in patients with castration-sensitive PC (CSPC) and BMs is also under discussion, as clinical trials with ZA in these patients have not shown significant benefits. Clinical data on the use of denosumab in CSPC are urgently needed

Circulating tumor cells and their role in prostate cancer

Circulating tumor cells (CTC) have become an important biomarker in patients with advanced prostate cancer. CTC count has been demonstrated to be a prognostic factor for overall survival in patients with metastatic castration-resistant prostate cancer (mCRPC). In localized prostate cancer, a clear correlation between CTC counts and clinicopathological risk parameters and outcome has not been observed. Currently, the focus of research is shifting from CTC enumeration towards molecular characterization of CTC leading to the discovery of markers predicting treatment response. The role of androgen receptor splice variants expressed by CTC as markers of resistance to abiraterone and enzalutamide has been assessed by various studies. The identification of CTC markers predicting treatment response represents a key step to guide the selection of treatment (e.g., abiraterone/enzalutamide vs taxanes), particularly in patients with mCRPC. As an alternative to CTC, the analysis of circulating tumor DNA has been shown to enable a noninvasive disease characterization having high potential to promote precision oncology