Development and characterization of the novel human osteosarcoma cell line COS-33 with sustained activation of the mTOR pathway.

Outcomes have not improved for metastatic osteosarcoma for several decades. In part, this failure to develop better therapies stems from a lack of understanding of osteosarcoma biology, given the rarity of the disease and the high genetic heterogeneity at the time of diagnosis. We report here the successful establishment of a new human osteosarcoma cell line, COS-33, from a patient-derived xenograft and demonstrate retention of the biological features of the original tumor. We found high mTOR signaling activity in the cultured cells, which were sensitive to a small molecule inhibitor, rapamycin, a suppressor of the mTOR pathway. Suppressed mTOR signaling after treatment with rapamycin was confirmed by decreased phosphorylation of the S6 ribosomal protein. Increasing concentrations of rapamycin progressively inhibited cell proliferation in vitro. We observed significant inhibitory effects of the drug on cell migration, invasion, and colony formation in the cultured cells. Furthermore, we found that only a strong osteogenic inducer, bone morphogenetic protein-2, promoted the cells to differentiate into mature mineralizing osteoblasts, indicating that the COS-33 cell line may have impaired osteoblast differentiation. Grafted COS-33 cells exhibited features typical of osteosarcoma, such as production of osteoid and tumorigenicity in vivo. In addition, we revealed that the COS-33 cell line retained a complex karyotype, a homozygous deletion of the TP53 gene, and typical histological features from its original tumor. Our novel cellular model may provide a valuable platform for studying the etiology and molecular pathogenesis of osteosarcoma as well as for testing novel drugs for future genome-informed targeted therapy.

The Management of Diabetes in Everyday Life (MODEL) program: development of a tailored text message intervention to improve diabetes self-care activities among underserved African-American adults.

Tailoring health-related materials is an effective mechanism to encourage behavior change; however, little research has described processes and critical characteristics for effective tailoring in underserved populations. The purpose of this study is to describe a process using input from content experts and lay patient advisors to tailor text messages focused on improving self-care behaviors of African-American adults with diabetes and identify characteristics of messages perceived to be most effective. An initial library of tailorable messages was created using theory-based approaches, expert opinion, and publicly available materials. A study-specific advisory council representing the program's intended population provided sequential individual and focus group review of a sample of draft messages focused on medication use, healthy eating, and physical activity. Messages were reviewed for content, tone, and applicability to African-American adults with diabetes from underserved communities. Based on this feedback, messages were revised and a final library of tailorable messages was constructed for use in a text messaging intervention. The initial library had over 5,000 tailorable messages. Participants preferred messages that included: (1) encouraging statements without condescension; (2) short sentences in lay language; (3) specific, actionable instructions; and (4) content relatable to daily activities of living. When possible, messages with similar themes should be repeated over short periods of time to improve the odds of material being absorbed and action being taken. Input from patient participants and advisors is essential for designing deeply tailored messages that honor the preferences, values, and norms of the population under study and promote health behavior change. TRIAL REGISTRATION: NCT02957513.

Generation of a CLTA reporter human induced pluripotent stem cell line, CRMi001-A-1, using the CRISPR/Cas9 system to monitor endogenous clathrin trafficking.

The most highly studied endocytic pathway, clathrin-dependent endocytosis, mediates a wide range of fundamental processes including nutrient internalization, receptor recycling, and signal transduction. In order to model tissue specific and developmental aspects of this process, CRISPR/Cas9 genomic editing was utilized to fluorescently label the C-terminus of clathrin light chain A (CLTA) within the phenotypically normal, parental CRMi001-A human induced pluripotent stem cell line. Successfully edited cells were isolated by fluorescently activated cell sorting, remained karyotypically normal, and maintained their differentiation potential. This cell line facilitates imaging of endogenous clathrin trafficking within varied cell types.

Comparative performance of isometric and isotonic quadriceps strength testing in total knee arthroplasty.

BACKGROUND: Early quadriceps muscle strength assessment after a total knee arthroplasty (TKA) provides timely information on progress, but little is known about the pain profile and predictive validity associated with common clinical muscle strength tests. This study aimed to, in patients with a recent TKA, examine the associations of isometric and isotonic quadriceps strength with gait speed, accounting for knee pain experienced during testing. METHODS: A sample of 76 patients (mean age 68 years; 46 women) with a recent TKA (median, 1.5 months) participated. Quadriceps strength was measured on both limbs using a knee extension machine. Isotonic strength was assessed with a one-repetition maximum test. Isometric strength was measured at 40° and 70° of knee flexion using a custom-built load cell. To allow for valid comparisons between the tests, quadriceps strength symmetry ratios were calculated. Knee pain during testing was measured using an 11-point pain scale. Fast gait speed was measured using the 10-m walk test. RESULTS: Compared with isotonic test, quadriceps strength ratio was higher for the 40° flexion isometric test (P = 0.01), and this difference may be explained by the lower knee pain intensity elicited during the isometric tests (P's < 0.001). All strength measures were closely associated with fast gait speed after adjustment for knee pain and covariates (P's < 0.001). CONCLUSIONS: Early in the post-TKA period, isometric and isotonic strength tests may be used to assess quadriceps strength but these tests are not interchangeable. Isometric quadriceps testing may be preferable to isotonic testing as it was associated with lower knee pain intensity.

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD.

Whole blood gene expression profiling of neonates with confirmed bacterial sepsis.

Neonatal infection remains a primary cause of infant morbidity and mortality worldwide and yet our understanding of how human neonates respond to infection remains incomplete. Changes in host gene expression in response to infection may occur in any part of the body, with the continuous interaction between blood and tissues allowing blood cells to act as biosensors for the changes. In this study we have used whole blood transcriptome profiling to systematically identify signatures and the pathway biology underlying the pathogenesis of neonatal infection. Blood samples were collected from neonates at the first clinical signs of suspected sepsis alongside age matched healthy control subjects. Here we report a detailed description of the study design, including clinical data collected, experimental methods used and data analysis workflows and which correspond with data in Gene Expression Omnibus (GEO) data sets (GSE25504). Our data set has allowed identification of a patient invariant 52-gene classifier that predicts bacterial infection with high accuracy and lays the foundation for advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.

Identification of a human neonatal immune-metabolic network associated with bacterial infection.

Understanding how human neonates respond to infection remains incomplete. Here, a system-level investigation of neonatal systemic responses to infection shows a surprisingly strong but unbalanced homeostatic immune response; developing an elevated set-point of myeloid regulatory signalling and sugar-lipid metabolism with concomitant inhibition of lymphoid responses. Innate immune-negative feedback opposes innate immune activation while suppression of T-cell co-stimulation is coincident with selective upregulation of CD85 co-inhibitory pathways. By deriving modules of co-expressed RNAs, we identify a limited set of networks associated with bacterial infection that exhibit high levels of inter-patient variability. Whereas, by integrating immune and metabolic pathways, we infer a patient-invariant 52-gene-classifier that predicts bacterial infection with high accuracy using a new independent patient population. This is further shown to have predictive value in identifying infection in suspected cases with blood culture-negative tests. Our results lay the foundation for future translation of host pathways in advancing diagnostic, prognostic and therapeutic strategies for neonatal sepsis.

Approaches to proximal tibial bone harvest techniques.

OBJECTIVES: The iliac crest is the standard site for harvesting bone; however, this procedure may require another specialist and a general anaesthetic. The proximal tibial bone harvest has gained popularity for harvesting autogenous bone. An analysis of the clinical literature regarding the various regions for harvesting bone demonstrates that the use of the proximal tibia led to shorter hospital stays, lower morbidity rates, and a shorter learning curve for the surgeon. The purpose of this study was to analyze the clinical anatomy of a proximal tibial bone harvest graft to provide the anatomical architecture supporting a safe procedure. MATERIALS AND METHODS: Dissection of 58 lower limbs from embalmed cadavers was conducted to determine the anatomy of a proximal tibial bone harvest (PTBH). RESULTS: Dissection revealed that the medial approach has fewer clinically relevant neurovascular structures in harms way, and a larger surface area, providing the clinician a confident surgical window to perform the procedure. CONCLUSIONS: The anatomical basis of this study suggests that the medial proximal tibial bone harvest approach would have fewer serious structures in harm's way compared to the lateral; however, the lateral approach may be preferred for a subgroup of patients.

Fast DNA and protein microarray tests for the diagnosis of hepatitis C virus infection on a single platform.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver cancer, and remains a large health care burden to the world. In this study we developed a DNA microarray test to detect HCV RNA and a protein microarray to detect human anti-HCV antibodies on a single platform. A main focus of this study was to evaluate possibilities to reduce the assay time, as a short time-to-result (TTR) is a prerequisite for a point-of-care test. Significantly reducing hybridisation and washing times did not impair the assay performance. This was confirmed first using artificial targets and subsequently using clinical samples from an HCV seroconversion panel derived from a HCV-infected patient. We were able to reduce the time required for the detection of human anti-HCV antibodies to only 14 min, achieving nanomolar sensitivity. The protein microarray exhibited an analytical sensitivity comparable to that of commercial systems. Similar results were obtained with the DNA microarray using a universal probe which covered all different HCV genotypes. It was possible to reduce the assay time after PCR from 150 min to 16 min without any loss of sensitivity. Taken together, these results constitute a significant step forward in the design of rapid, microarray-based diagnostics for human infectious disease, and show that the protein microarray is currently the most favourable candidate to fill this role.

Proteolysis-inducing factor core peptide mediates dermcidin-induced proliferation of hepatic cells through multiple signalling networks.

Dermcidin is a candidate oncogene capable of increasing the number of cultured neuronal, breast cancer and prostate cancer cells and improving the survival of hepatic cells. The dermcidin gene encodes the proteolysis-inducing factor core peptide (PIF-CP) and the skin antimicrobial peptide DCD-1. The peptide responsible for inducing proliferation of cells and the mechanisms involved are unknown. In this study, we confirmed a proliferative effect of dermcidin overexpression of 20% (p<0.02) in the HuH7 human hepatic cell line. Proliferation was abrogated by prevention of PIF-CP translation or inactivation of its calcineurin-like phosphatase domain by site-directed mutagenesis. Prevention of DCD-1 translation had no effect. Treatment of cells with a 30 amino acid synthetic PIF-CP induced an analogous increase in proliferation of 14%. Microarray analysis of PIF-CP-treated cells revealed low but significant changes in 111 potential mediator genes. Pathway analysis revealed several gene networks involved in the cellular response to the peptide, one with VEGFB as a hub and two other networks converging on FOS and MYC. Quantitative PCR confirmed direct upregulation of VEGFB. These data reveal PIF-CP as the key mediator of dermcidin-induced proliferation and demonstrate induction of key oncogenic pathways.

Temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes.

The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.

Peptide-tags for enhanced DNA microarray performance.

DNA microarrays are powerful tools for gene expression analysis and genotyping studies in research and diagnostic applications. A high sensitivity and short time-to-result are prerequisites for their practical application in the clinic. The hybridization efficiency of DNA microarrays depends on the probe density and the probe orientation and thus their accessibility for target molecules. In order to find an optimal probe immobilization procedure a set of different oligonucleotide modifications was tested on epoxy silane functionalized glass slides. It was found that histidine-tagged oligonucleotides resulted in the highest amount of bound probe and by far the best hybridization efficiencies. The detection limit obtained with histidine-tagged probes was up to two orders of magnitude lower compared to commonly used probe modifications. In order to further investigate the binding mechanism of histidine-tags towards functionalized glass substrates a set of different peptide-tags with and without free terminal amino-groups and with different amino acid compositions was tested. The results indicate an impact of the terminal amino group on the covalent surface binding and of aromatic amino acid residues on the enhanced hybridisation efficiency.

Multi-factorial analysis of class prediction error: estimating optimal number of biomarkers for various classification rules.

Machine learning and statistical model based classifiers have increasingly been used with more complex and high dimensional biological data obtained from high-throughput technologies. Understanding the impact of various factors associated with large and complex microarray datasets on the predictive performance of classifiers is computationally intensive, under investigated, yet vital in determining the optimal number of biomarkers for various classification purposes aimed towards improved detection, diagnosis, and therapeutic monitoring of diseases. We investigate the impact of microarray based data characteristics on the predictive performance for various classification rules using simulation studies. Our investigation using Random Forest, Support Vector Machines, Linear Discriminant Analysis and k-Nearest Neighbour shows that the predictive performance of classifiers is strongly influenced by training set size, biological and technical variability, replication, fold change and correlation between biomarkers. Optimal number of biomarkers for a classification problem should therefore be estimated taking account of the impact of all these factors. A database of average generalization errors is built for various combinations of these factors. The database of generalization errors can be used for estimating the optimal number of biomarkers for given levels of predictive accuracy as a function of these factors. Examples show that curves from actual biological data resemble that of simulated data with corresponding levels of data characteristics. An R package optBiomarker implementing the method is freely available for academic use from the Comprehensive R Archive Network (http://www.cran.r-project.org/web/packages/optBiomarker/).

Longitudinal assessment of lung cancer progression in the mouse using in vivo micro-CT imaging.

PURPOSE: Small animal micro-CT imaging is being used increasingly in preclinical biomedical research to provide phenotypic descriptions of genomic models. Most of this imaging is coincident with animal death and is used to show the extent of disease as an end point. Longitudinal imaging overcomes the limitation of single time-point imaging because it enables tracking of the natural history of disease and provides qualitative and, where possible, quantitative assessments of the effects of an intervention. The pulmonary system is affected by many disease conditions, such as lung cancer, chronic obstructive pulmonary disease, asthma, and granulomatous disorders. Noninvasive imaging can accurately assess the lung phenotype within the living animal, evaluating not only global lung measures, but also regional pathology. However, imaging the lung in the living animal is complicated by rapid respiratory motion, which leads to image based artifacts. Furthermore, no standard mouse lung imaging protocols exist for longitudinal assessment, with each group needing to develop their own systematic approach. METHODS: In this article, the authors present an outline for performing longitudinal breath-hold gated micro-CT imaging for the assessment of lung nodules in a mouse model of lung cancer. The authors describe modifications to the previously published intermittent isopressure breath-hold technique including a new animal preparation and anesthesia protocol, implementation of a ring artifact reduction, variable scanner geometry, and polynomial beam hardening correction. In addition, the authors describe a multitime-point data set registration and tumor labeling and tracking strategy. RESULTS: In vivo micro-CT data sets were acquired at months 2, 3, and 4 posturethane administration in cancer mice (n = 5) and simultaneously in control mice (n = 3). 137 unique lung nodules were identified from the cancer mice while no nodules were detected in the control mice. A total of 411 nodules were segmented and labeled over the three time-points. Lung nodule metrics including RECIST, Ortho, WHO, and 3D volume were determined and extracted. A tumor incidence rate of 30.44 +/- 1.93 SEM for n = 5 was found with identification of nodules as small as 0.11 mm (RECIST) and as large as 1.66 mm (RECIST). In addition, the tumor growth and doubling rate between months 2-3 and 3-4 were calculated. Here, the growth rate was slightly higher in the second period based on the 3D volume data (0.12 +/- 0.13 to 0.13 +/- 0.17 microl) but significantly less based on the linear diameter metrics [RECIST (0.33 +/- 0.19 to 0.17 +/- 0.18 mm); Ortho (0.24 +/- 0.15 to 0.16 +/- 0.15 mm)], indicating the need to understand how each metric is obtained and how to correctly interpret change in tumor size. CONCLUSIONS: In conclusion, micro-CT imaging provides a unique platform for in vivo longitudinal assessment of pulmonary lung cancer progression and potentially tracking of therapies at very high resolutions. The ability to evaluate the same subject over time provides for a sensitive assay that can be carried out on a smaller sample size. When integrated with image processing and analysis routines as detailed in this study, the data acquired from micro-CT imaging can now provide a very powerful assessment of pulmonary disease outcomes.

Assessment of transcriptomal analysis of Varicella-Zoster-virus gene expression in patients with and without post-herpetic neuralgia.

Varicella-Zoster virus (VZV) is a human herpes virus that reactivates from a latent state in human trigeminal and dorsal root ganglia to cause herpes zoster (shingles) which is a painful vesicular dermatomal skin eruption. The major complication of herpes zoster is post-herpetic neuralgia (PHN) which is a serious condition occurring especially in individuals over 50 years. PHN is extremely painful, may be permanent, and is frequently very refractory to all treatment. The ability to identify those patients with herpes zoster who are likely to develop PHN would be highly beneficial as it would allow pre-emptive anti-viral therapy. We have assessed the potential of using long oligonucleotide VZV microarrays to determine whether MeWo cells infected with VZV isolates obtained from 13 patients with zoster who had subsequently developed PHN showed significant transcriptomal differences from MeWo cells infected with viruses isolated from ten zoster patients who had not developed PHN. We found that viral gene expression from sample to sample within a group (PHN patients or non-PHN patients) varied as much, or more, than the viral gene expression between those groups. Quantitative real-time polymerase chain reaction studies carried out on 11 open reading frames on four representative viral infected MeWo cell lines (two from each group) confirmed the transcriptomal heterogeneity between the two groups. Growth curve analyses of ten representative infected cell lines (five from each group) showed that PHN and non-PHN-associated viruses replicated equally efficiently. Taken together, these findings suggest that viral microarray-based transcriptomal measurements are unlikely to prove of clinical utility in predicting the incidence of PHN.

Training Outcomes from the Samaritans of New York Suicide Awareness and Prevention Programme Among Community- and School-based Staff.

The Samaritans of New York public education suicide awareness and prevention programme is designed to train lay and professional staff on effective suicide prevention practices and how to "befriend" a person in crisis. However, little is known about the individual level characteristics of staff who attend these trainings. Community- and school-based staff (N=365) completed pre and post training measures of self-efficacy regarding their knowledge about suicide and suicide prevention and their ability to intervene with individuals at risk for suicide. Results indicate increased self-efficacy after suicide prevention training (M=3.7, SD=0.6) than before (M=3.3, SD=0.7) (t= -13.24, p<.05). Trainees with higher levels of education and previous contact with suicidal individuals were significantly more likely to indicate gains in self-efficacy after training.

Pulmonary hypertension in thoracic surgical patients.

PURPOSE OF REVIEW: Literature about thoracic surgery in patients with pulmonary hypertension is scarce. Perceived high risk has appropriately discouraged any unnecessary operation. However, the medical therapy for pulmonary hypertension has made great advances during the last decade. It is likely that future advances in survival and possibly the need for diagnostic procedures will increase the anesthesiologist's exposure to such patients. Understanding the unique physiology as well as new therapeutic agents will facilitate safe care for these challenging patients. RECENT FINDINGS: Since 1998, there have been three World Heath Organization symposiums on pulmonary hypertension. The most recent meeting in 2008 at Dana Point included revisions of the classification scheme and updates on new trials and therapies. New drugs have been utilized in cardiac, lung, or liver transplant operations to treat pulmonary hypertension. It is also recognized that one-lung ventilation presents unique problems for the patient with pulmonary hypertension. Inhalation use of the new pulmonary vasodilator drugs represents a new frontier for intraoperative pharmacology. SUMMARY: Here, the various types of pulmonary hypertension, physiologic changes, and new drug therapies are reviewed. Clinical experience with patients with pulmonary hypertension undergoing both nonthoracic and thoracic procedures is also reviewed. By identifying potential problem areas and application of new pharmacology, an approach to the patient with pulmonary hypertension is synthesized.

Genome-wide expression profiling of in vivo-derived bloodstream parasite stages and dynamic analysis of mRNA alterations during synchronous differentiation in Trypanosoma brucei.

BACKGROUND: Trypanosomes undergo extensive developmental changes during their complex life cycle. Crucial among these is the transition between slender and stumpy bloodstream forms and, thereafter, the differentiation from stumpy to tsetse-midgut procyclic forms. These developmental events are highly regulated, temporally reproducible and accompanied by expression changes mediated almost exclusively at the post-transcriptional level. RESULTS: In this study we have examined, by whole-genome microarray analysis, the mRNA abundance of genes in slender and stumpy forms of T.brucei AnTat1.1 cells, and also during their synchronous differentiation to procyclic forms. In total, five biological replicates representing the differentiation of matched parasite populations derived from five individual mouse infections were assayed, with RNAs being derived at key biological time points during the time course of their synchronous differentiation to procyclic forms. Importantly, the biological context of these mRNA profiles was established by assaying the coincident cellular events in each population (surface antigen exchange, morphological restructuring, cell cycle re-entry), thereby linking the observed gene expression changes to the well-established framework of trypanosome differentiation. CONCLUSION: Using stringent statistical analysis and validation of the derived profiles against experimentally-predicted gene expression and phenotypic changes, we have established the profile of regulated gene expression during these important life-cycle transitions. The highly synchronous nature of differentiation between stumpy and procyclic forms also means that these studies of mRNA profiles are directly relevant to the changes in mRNA abundance within individual cells during this well-characterised developmental transition.