16 research outputs found
Amino Acid Quenching
Alexa Fluor 594 is a fluorescent molecule commonly used to tag calmodulin, a protein that regulates a variety of cell processes. By measuring fluorescence, scientists can study calmodulin and its interactions with other proteins. However, fluorescence is not the only fate of Alexa Fluor 594. The molecule is also known to transfer energy to coupled amino acids through a mechanism called quenching. The purpose of this experiment was to study the quenching effects of Histidine, Tyrosine, and Methionine on the Alexa Fluor 594. These amino acidsâ Stern-Volmer plots suggest static quenching may be occurring. Moreover, the Stern-Volmer plot of Potassium Iodide (KI), a dynamic quencher, suggested that KI quenches the excitation Alexa Fluor 594 in part through a static mechanism. We plan to replicate our experiment and complete statistical testing to determine the significance of our data. Determining if these amino acids quench AlexaFluor594 will help researchers better characterize the calmodulin protein and its interactions
Exploring Gene Functions and Phage-Host Protein Interactions in Mycobacteriophage Island3
Island3 is an I1 mycobacteriophage that infects Mycobacterium smegmatis mcÂČ155. It has a total of 76 protein coding genes, but only 17 of these genes have functions assigned by bioinformatics. To discover the functions of the additional genes, we cloned 72 of Island3âs genes and are assaying each gene product for two functions when expressed in the host M. smegmatis: the ability to reduce growth of the host (cytotoxicity) and the ability to protect the host from infection by Island3 or another phage (defense). So far, we have assayed more than 60 of Island3âs genes and found 14 genes that exhibited cytotoxicity but none that exhibited definitive defense against phage infection. We are currently analyzing the remaining genes for cytotoxicity and defense. In addition, we are moving forward with bacterial two-hybrid assays on two of the genes that exhibited cytotoxicity, seeking to identify host proteins that interact with the cytotoxic phage gene products in an attempt to understand the mechanism of cytotoxicity.
The association between preoperative body composition and aerobic fitness in patients scheduled for colorectal surgery
AIM: Although cardiopulmonary exercise testing (CPET) is considered the gold standard, a preoperative abdominal CT scan might also provide information concerning preoperative aerobic fitness for risk assessment. This study aimed to investigate the association between preoperative CTâscanâderived body composition variables and preoperative CPET variables of aerobic fitness in colorectal surgery. METHOD: In this retrospective cohort study, CT images at level L3 were analysed for skeletal muscle mass, skeletal muscle radiation attenuation, visceral adipose tissue (VAT) mass and subcutaneous adipose tissue mass. Regression analyses were performed to investigate the relation between CTâscanâderived body composition variables, CPETâderived aerobic fitness and other preoperative patientârelated variables. Logistic regression analysis was performed to predict a preoperative anaerobic threshold (AT) â€Â 11.1 ml/kg/min as cutâoff for having a high risk for postoperative complications. RESULTS: Data from 78 patients (45 men; mean [SD] age 74.5 [6.4 years]) were analysed. A correlation coefficient of 0.55 was observed between absolute AT and skeletal muscle mass index. Absolute AT (R (2) of 51.1%) was lower in patients with a lower skeletal muscle mass index, together with higher age, lower body mass and higher American Society of Anesthesiologists (ASA) score. Higher ASA score (odds ratio 5.64; P = 0.033) and higher VAT mass (odds ratio 1.02; P = 0.036) were associated with an increased risk of an AT â€Â 11.1 ml/kg/min. CONCLUSION: Body composition variables from the preoperative CT scan were moderately associated with preoperative CPETâderived aerobic fitness. Higher ASA score and higher VAT mass were associated with an increased risk of an AT â€Â 11.1 ml/kg/min
Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas
Medulloblastoma is the most common malignant brain tumor in childhood. Molecular studies from several groups around the world demonstrated that medulloblastoma is not one disease but comprises a collection of distinct molecular subgroups. However, all these studies reported on different numbers of subgroups. The current consensus is that there are only four core subgroups, which should be termed WNT, SHH, Group 3 and Group 4. Based on this, we performed a meta-analysis of all molecular and clinical data of 550 medulloblastomas brought together from seven independent studies. All cases were analyzed by gene expression profiling and for most cases SNP or array-CGH data were available. Data are presented for all medulloblastomas together and for each subgroup separately. For validation purposes, we compared the results of this meta-analysis with another large medulloblastoma cohort (n = 402) for which subgroup information was obtained by immunohistochemistry. Results from both cohorts are highly similar and show how distinct the molecular subtypes are with respect to their transcriptome, DNA copy-number aberrations, demographics, and survival. Results from these analyses will form the basis for prospective multi-center studies and will have an impact on how the different subgroups of medulloblastoma will be treated in the future
Integrated Genomics Identifies Five Medulloblastoma Subtypes with Distinct Genetic Profiles, Pathway Signatures and Clinicopathological Features
BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms. METHODS AND FINDINGS: To get a better insight into the molecular biology of medulloblastoma we established mRNA expression profiles of 62 medulloblastomas and analyzed 52 of them also by comparative genomic hybridization (CGH) arrays. Five molecular subtypes were identified, characterized by WNT signaling (A; 9 cases), SHH signaling (B; 15 cases), expression of neuronal differentiation genes (C and D; 16 and 11 cases, respectively) or photoreceptor genes (D and E; both 11 cases). Mutations in beta-catenin were identified in all 9 type A tumors, but not in any other tumor. PTCH1 mutations were exclusively identified in type B tumors. CGH analysis identified several fully or partly subtype-specific chromosomal aberrations. Monosomy of chromosome 6 occurred only in type A tumors, loss of 9q mostly occurred in type B tumors, whereas chromosome 17 aberrations, most common in medulloblastoma, were strongly associated with type C or D tumors. Loss of the inactivated X-chromosome was highly specific for female cases of type C, D and E tumors. Gene expression levels faithfully reflected the chromosomal copy number changes. Clinicopathological features significantly different between the 5 subtypes included metastatic disease and age at diagnosis and histology. Metastatic disease at diagnosis was significantly associated with subtypes C and D and most strongly with subtype E. Patients below 3 yrs of age had type B, D, or E tumors. Type B included most desmoplastic cases. We validated and confirmed the molecular subtypes and their associated clinicopathological features with expression data from a second independent series of 46 medulloblastomas. CONCLUSIONS: The new medulloblastoma classification presented in this study will greatly enhance the understanding of this heterogeneous disease. It will enable a better selection and evaluation of patients in clinical trials, and it will support the development of new molecular targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life
Sphingomyelin Functions as a Novel Receptor for Helicobacter pylori VacA
The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells
Advocacy for use of the modified Iowa Level of Assistance Scale for clinical use in patients after hip replacement: an observational study
Objectives: o test the internal consistency and item difficulty of the modified Iowa Level of Assistance Scale (mILAS). Design: Retrospective observational study. Setting: Two orthopaedic wards of two general hospitals. Participants"Following elective primary unilateral total hip replacement surgery, all participants performed mILAS activities that were scored daily to assess their recovery of activities during hospitalisation. Main outcome measures: The internal consistency and the level of assistance needed by the patient (item difficulty) of the mILAS were calculated using data from Hospital X (nâ
=â
255). A cross-validation was performed using data from Hospital Y (nâ
=â
224). Results: The internal consistency of the mILAS was acceptable on all three postoperative days (α=0.84 to 0.97). Cronbachâs α and Rasch analysis revealed a misfit of stair climbing with the other items of the mILAS. The item difficulty of the mILAS items changed over the first two postoperative days. During the first three postoperative days, the sit to supine transfer was generally the most difficult item to achieve, and the sit to stand transfer was the least difficult item to achieve as rated by physiotherapists. The cross-validation analysis revealed similar results. Conclusions: The mILAS is a clinically sound measurement tool to assess the ability of patients to perform five functional tasks safely during hospitalisation. Stair climbing appears to be the easiest item to complete, and the sit to supine transfer is generally the most difficult after surgery
The association between preoperative body composition and aerobic fitness in patients scheduled for colorectal surgery
Aim Although cardiopulmonary exercise testing (CPET) is considered the gold standard, a preoperative abdominal CT scan might also provide information concerning preoperative aerobic fitness for risk assessment. This study aimed to investigate the association between preoperative CT-scan-derived body composition variables and preoperative CPET variables of aerobic fitness in colorectal surgery. Method In this retrospective cohort study, CT images at level L3 were analysed for skeletal muscle mass, skeletal muscle radiation attenuation, visceral adipose tissue (VAT) mass and subcutaneous adipose tissue mass. Regression analyses were performed to investigate the relation between CT-scan-derived body composition variables, CPET-derived aerobic fitness and other preoperative patient-related variables. Logistic regression analysis was performed to predict a preoperative anaerobic threshold (AT) â€Â 11.1 ml/kg/min as cut-off for having a high risk for postoperative complications. Results Data from 78 patients (45 men; mean [SD] age 74.5 [6.4 years]) were analysed. A correlation coefficient of 0.55 was observed between absolute AT and skeletal muscle mass index. Absolute AT (R2 of 51.1%) was lower in patients with a lower skeletal muscle mass index, together with higher age, lower body mass and higher American Society of Anesthesiologists (ASA) score. Higher ASA score (odds ratio 5.64; P = 0.033) and higher VAT mass (odds ratio 1.02; P = 0.036) were associated with an increased risk of an AT â€Â 11.1 ml/kg/min. Conclusion Body composition variables from the preoperative CT scan were moderately associated with preoperative CPET-derived aerobic fitness. Higher ASA score and higher VAT mass were associated with an increased risk of an AT â€Â 11.1 ml/kg/min