Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Flexible Multibody Simulation Approach in the Dynamic Analysis of Bone Strains Activity

The objective of this study is to show that bone strains due to dynamic mechanical loading during physical activity can be analysed using the flexible multibody simulation approach. Strains within the bone tissue play a major role in bone (re)modeling. Based on previous studies, it has been shown that dynamic loading seems to be more important for bone (re)modeling than static loading. The finite element method has been used previously to assess bone strains. However, the finite element method may be limited to static analysis of bone strains due to the expensive computation required for dynamic analysis, especially for a biomechanical system consisting of several bodies. Further, in vivo implementation of strain gauges on the surfaces of bone has been used previously in order to quantify the mechanical loading environment of the skeleton. However, in vivo strain measurement requires invasive methodology, which is challenging and limited to certain regions of superficial bones only, such as the anterior surface of the tibia. In this study, an alternative numerical approach to analyzing in vivo strains, based on the flexible multibody simulation approach, is proposed. In order to investigate the reliability of the proposed approach, three 3-dimensional musculoskeletal models where the right tibia is assumed to be flexible, are used as demonstration examples. The models are employed in a forward dynamics simulation in order to predict the tibial strains during walking on a level exercise. The flexible tibial model is developed using the actual geometry of the subject’s tibia, which is obtained from 3 dimensional reconstruction of Magnetic Resonance Images. Inverse dynamics simulation based on motion capture data obtained from walking at a constant velocity is used to calculate the desired contraction trajectory for each muscle. In the forward dynamics simulation, a proportional derivative servo controller is used to calculate each muscle force required to reproduce the motion, based on the desired muscle contraction trajectory obtained from the inverse dynamics simulation. Experimental measurements are used to verify the models and check the accuracy of the models in replicating the realistic mechanical loading environment measured from the walking test. The predicted strain results by the models show consistency with literature-based in vivo strain measurements. In conclusion, the non-invasive flexible multibody simulation approach may be used as a surrogate for experimental bone strain measurement, and thus be of use in detailed strain estimation of bones in different applications. Consequently, the information obtained from the present approach might be useful in clinical applications, including optimizing implant design and devising exercises to prevent bone fragility, accelerate fracture healing and reduce osteoporotic bone loss.

Postoperative Adjuvant Lapatinib and Concurrent Chemoradiotherapy Followed by Maintenance Lapatinib Monotherapy in High-Risk Patients With Resected Squamous Cell Carcinoma of the Head and Neck: A Phase III, Randomized, Double-Blind, Placebo-Controlled Study.

PURPOSE: This multicenter phase III study evaluated the efficacy and safety of lapatinib, an epidermal growth factor receptor/ErbB2 inhibitor, administered concomitantly with chemoradiotherapy and as maintenance monotherapy in patients with high-risk surgically treated squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Patients with resected stage II to IVA SCCHN, with a surgical margin ≤ 5 mm and/or extracapsular extension, were randomly assigned to chemoradiotherapy (66 Gy total radiation dose and cisplatin 100 mg/m(2) per day administered on days 1, 22, and 43) plus placebo or lapatinib (1,500 mg per day) before and during chemoradiotherapy, followed by 12 months of maintenance monotherapy. RESULTS: Six hundred eighty-eight patients were enrolled (lapatinib, n = 346; placebo, n = 342). With a median follow-up time of 35.3 months, the study ended early because of the apparent plateauing of disease-free survival (DFS) events. Median DFS assessed by an independent review committee was 53.6 months and not reached for lapatinib and placebo, respectively (hazard ratio, 1.10; 95% CI, 0.85 to 1.43). Investigator-assessed results confirmed the independent review committee assessment. No significant differences in DFS by human papillomavirus status or overall survival were observed between treatment arms. Similar numbers of patients in both treatment arms experienced adverse events (AEs), with more patients in the lapatinib arm than the placebo arm experiencing serious AEs (48% v 40%, respectively). The most commonly observed treatment-related AEs were diarrhea and rash, both predominantly in the lapatinib arm. CONCLUSION: Addition of lapatinib to chemoradiotherapy and its use as long-term maintenance therapy does not offer any efficacy benefits and had additional toxicity compared with placebo in patients with surgically treated high-risk SCCHN.

Magnetic resonance imaging and BMB score in the evaluation of bone involvement in Gaucher’s disease patients

AbstractObjective:To evaluate by magnetic resonance imaging changes in bone marrow of patients undergoing treatment for type I Gaucher’s disease.Materials and Methods:Descriptive, cross-sectional study of Gaucher’s disease patients submitted to 3 T magnetic resonance imaging of femurs and lumbar spine. The images were blindly reviewed and the findings were classified according to the semiquantitative bone marrow burden (BMB) scoring system.Results:All of the seven evaluated patients (three men and four women) presented signs of bone marrow infiltration. Osteonecrosis of the femoral head was found in three patients, Erlenmeyer flask deformity in five, and no patient had vertebral body collapse. The mean BMB score was 11, ranging from 9 to 14.Conclusion:Magnetic resonance imaging is currently the method of choice for assessing bone involvement in Gaucher’s disease in adults due to its high sensitivity to detect both focal and diffuse bone marrow changes, and the BMB score is a simplified method for semiquantitative analysis, without depending on advanced sequences or sophisticated hardware, allowing for the classification of the disease extent and assisting in the treatment monitoring.

Callus cell culture of Pothomorphe umbellata (L.) under stress condition leads to high content of peroxidase enzyme

Pothomorphe umbellata (L.) known on Brazil as Caapeba has a number of popular medicinal use, and it has been studied in relation to its pharmacological activity. Peroxidase specific activity (units/mg protein) was evaluated in callus cell culture samples of the P.umbellata, grown in two different MS medium (media 1 and media 2), submitted to 16 hours photoperiod or kept in darkness. Cell growth rate curve showed that the best growth indices were observed when media 2 submitted to the photoperiod regime was used, followed by the same media kept in darkness (stress condition). The results obtained also showed that the cell culture grown under stress conditions (darkness) lead to high content of peroxidase enzyme (an increase of 700% was observed). Kinetic constant values of 3.3 mmol.L-1 and 2,8 sec-1 were obtained for kM and v max,, respectively, using guaiacol as enzyme substrate.

First-Line Chemotherapy with Anthracycline and Taxane Combination in Metastatic Breast Cancer. Detection of bone metastases with TRACP 5b

Background: In Finland, breast cancer (BC) is the most common cancer among women, and prostate cancer (PC) that among men. At the metastatic stage both cancers remain essentially incurable. The goals of therapy include palliation of symptoms, improvement or maintenance of quality of life (QoL), delay of disease progression, and prolongation of survival. Balancing between efficacy and toxicity is the major challenge. With increasing costs of new treatments, appropriate use of resources is paramount. When new treatment regimes are introduced into clinical practice a comprehensive assessment of clinical benefit, adverse effects and cost is necessary. Both BC and PC show a predilection to metastasize to bone. Bone metastases cause significant morbidity impairing the patients´ QoL. Diagnosis of bone metastases relies mainly on radiological methods, which however lack optimal sensitivity and specificity. New tools are needed for detection and follow-up of bone metastases. Aims: Anthracyclines and taxanes are effective chemotherapeutic agents in the treatment of metastatic breast cancer (MBC) with different mechanisms of action. Therefore, evaluation of the combination of anthracyclines with taxanes was a justifiable approach in the treatment of MBC patients. We assessed the efficacy, toxicity, cost of treatment and QoL of BC patients treated with first-line chemotherapy for metastatic disease with the combination epirubicin and docetaxel. We also evaluated the diagnostic potential of tartrate-resistant acid phosphatase 5b (TRACP 5b) and carboxyterminal telopeptides of type I collagen (ICTP) in the diagnosis of bone metastases in BC and TRACP 5b in PC patients. Results: The combination of epirubicin and docetaxel was effective in this phase II study, but required individual dose adjustment to avoid neutropenic infections, and the use of growth factors to maintain a feasible dose level. The response rate was 54 % (95 % CI 37-71) and the median overall survival (OS) was 26 months. Of the patients, 87 % were treated for infections. The treatment of adverse events required additional use of health resources mainly due to neutropenic infections, thereby raising direct treatment costs by 20 %. Despite adverse events, the global QoL was not significantly compromised during the treatment. Clinically evident acute cardiac toxicity was not observed. The combination of serum TRACP 5b and ICTP was at least equally sensitive and specific in detection of of bone metastases as commonly used total alkaline phosphatise (tALP) in BC patients. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC patients. Conclusions: Treatment with epirubicin and docetaxel showed high efficacy in first-line chemotherapy of MBC. The relatively high incidence of neutropenic infections requiring hospitalization increased the treatment costs. Despite adverse events, the global QoL of the patients was not significantly compromised. The combination of TRACP 5b and ICTP showed similar activity as tALP in detecting bone metastases in MBC. In contrast, TRACP 5b was less specific and sensitive than tALP as a marker of skeletal changes in PC.

Genetic polymorphisms in the Cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx

Objective:To analyze the genetic polymorphisms of the cytochrome P450 family and their relationship with squamous cell carcinoma of the oral cavity, pharynx and larynx.Methods: We present a narrative literature review, conducted in Pubmed, Lilacs and Cochrane Databases of articles published in the last five years correlating genetic polymorphisms of the cytochrome P450 family and cancer risk in different populations worldwide.Results: We initially found 65 articles and, after selection criteria, 20 case-control studies with various populations worldwide were eligible. The most studied polymorphisms were those of CYP2E1 and CYP1A1 subfamilies. There is little about the other subfamilies. The association found between polymorphisms and cancer risk amounted to a countless number of variables, amongst them: population, selection methods, racial factors and different modes of exposure to carcinogens, genotyping methods, and nomenclature of the polymorphisms.Conclusion: so far, there is no proven link between genetic polymorphisms of cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx relationship.

Importance of bone assessment and prevention of osteoporotic fracture in patients with prostate cancer in the gonadotropic hormone analogues use

The antiandrogenic therapy (ADT) for prostate cancer represents an additional risk factor for the development of osteoporosis and fragility fractures. Still, bone health of patients on ADT is often not evaluated. After literature research we found that simple preventive measures can prevent bone loss in these patients, resulting in more cost-effective solutions to the public health system and family when compared to the treatment of fractures.

Effect of bone morphogenetic protein-7 (BMP-7) on in vitro survival of caprine preantral follicles

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.