N-Myc oncogene amplification is a frequent event in neuroblastoma and is strongly correlated with advanced disease stage and treatment failure. Similarly to c-Myc oncogenic activation, N-Myc deregulation promotes both cell proliferation and p53-dependent apoptosis by sensitizing cells to a variety of insults. Intriguingly, p53 mutations are uncommon in neuroblastomas, strongly suggesting that an alternative cooperating event circumvents this safeguard against oncogene-driven neoplasia. By performing a pangenomic cDNA microarray analysis, we demonstrate that human Twist is constantly overexpressed in N-Myc-amplified neuroblastomas. H-Twist overexpression is responsible for the inhibition of the ARF/p53 pathway involved in the Myc-dependent apoptotic response. This oncogenic cooperation of two key regulators of embryogenesis causes cell transformation and malignant outgrowth.

Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from human endogenous retrovirus K (HERV-K) (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either reverse transcriptase PCR or nucleic acid sequence-based amplification. Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly processed HERV-K (HML-2) Gag and envelope proteins. Finally, using immunoelectron microscopy, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.

Whole blood transcriptional signatures distinguishing active tuberculosis patients from asymptomatic latently infected individuals exist. Consensus has not been achieved regarding the optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. Here we show a blood transcriptional signature of active tuberculosis using RNA-Seq, confirming microarray results, that discriminates active tuberculosis from latently infected and healthy individuals, validating this signature in an independent cohort. Using an advanced modular approach, we utilise the information from the entire transcriptome, which includes overabundance of type I interferon-inducible genes and underabundance of IFNG and TBX21, to develop a signature that discriminates active tuberculosis patients from latently infected individuals or those with acute viral and bacterial infections. We suggest that methods targeting gene selection across multiple discriminant modules can improve the development of diagnostic biomarkers with improved performance. Finally, utilising the modular approach, we demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.

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Whole blood transcriptional signatures distinguishing patients with active tuberculosis from asymptomatic latently infected individuals have been described but, no consensus exists for the composition of optimal reduced gene sets as diagnostic biomarkers that also achieve discrimination from other diseases. We have recapitulated a blood transcriptional signature of active tuberculosis using RNA-Seq, previously reported by microarray that discriminates active tuberculosis from latently infected and healthy individuals, also validated in an independent cohort. We show that an advanced modular approach, which preserves and presents a signature of the entire transcriptome, can better discriminate patients with active tuberculosis from both latently infected and acute viral and bacterial infections. We suggest a method of targeted gene selection across modules for constructing diagnostic biomarkers, more representative of the transcriptome that overcomes some limitations of existing techniques. Finally, we utilise the modular approach to demonstrate dynamic heterogeneity in a longitudinal study of recent tuberculosis contacts.Tuberculosis (TB) is the leading cause of global mortality from an infectious disease. In 2016, there were 6.3 million new cases of TB disease and 1.67 million deaths and its diagnosis is problematic 1 . However, clinical disease represents one end of a spectrum of infection states. It is estimated that up to one third of all individuals worldwide have been infected with the causative pathogen, Mycobacterium tuberculosis, but the vast majority remain clinically asymptomatic with no radiological or microbiological evidence for active infection. This is termed latent TB infection (LTBI) and conceptually denotes a state in which M. tuberculosis persists within its host, while maintaining viability with the potential to replicate and cause symptomatic disease. Indeed, LTBI represents the primary reservoir for future incident TB, with 90% of all TB cases estimated to arise from reactivation of existing infection 1,2 . The risk of incident TB arising from existing LTBI is heterogeneous, poorly characterised and modifiable with anti-tuberculous treatment. Modelling studies indicate effective TB prevention to significantly reduce future TB incidence requires policies directed at the identification and treatment of LTBI 3 . However, implementation of mass screening programmes for this purpose are severely constrained by the size of the target population. Transformative advances in diagnostic tools that can effectively stratify TB risk in the LTBI population are therefore implicit to the realisation of systematic screening.The basis for LTBI heterogeneity rests with the limited scope of the tools we have available to identify the state. LTBI is inferred solely through evidence that immune sensitization has occurred, by the tuberculin skin test (TST) or the M. tuberculosis antigen-specific interferon-gamma (IFN-g) release assay (IGRA). Although these tests are both sensitive and specific for identi...

This protocol describes a microfluidic platform for dynamic high-throughput analysis the phenotype of single cells. Cell-surface markers and secreted proteins are quantified and characterized by fluorescence detection using tailored immunoassays, simultaneously with measurement of other cellular characteristics, including endocytosis activity and viability.TWEET A new protocol describes a microfluidics-based assay for high-throughput interrogation of protein secretion kinetics in single cells. @BIOASTER @EyerFira @ETH_DCHAB COVER TEASER Microfluidics-based analysis of protein secretion Up to three primary research articles where the protocol has been used and/or developed.

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5 cytomegalovirus early promoter, the 5 viral sequences up to the 5 end of gag required for reverse transcription and packaging, the Rev-responsive element, a geneexpressing cassette, and the 3 long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10 Gene transfer with potential therapeutic applications represents a very important and exciting challenge in biology and medicine. To this end, several generations of retroviral vectors derived from type C retroviruses have been developed that are capable of delivering a gene into many different cell types. For example, MLV-based dicistronic vectors were recently found to efficiently deliver two genes in human multipotent neuronal precursors (9). However, an important limitation of murine leukemia virus (MLV)-based vectors is their inability to deliver a gene into highly differentiated or growth-arrested cells. On the other hand, MLV vectors can be used in cancer gene therapy (17).Interestingly enough, lentiviruses are able to infect growtharrested cells (19) and this appears to be under the control of genetic determinants located in the integrase domain and in the vpx gene encoding a highly basic protein. In support of these genetic findings, the integrase and Vpx proteins are present in the viral particle and are thought to drive the nuclear import of the reverse transcription complex, also called the preintegration complex, probably by interacting with components of the nuclear pore complex (13). This route of viral infection, which appears to be unique to lentiviruses in the retrovirus family, has been exploited for the development of lentivirus vectors derived from human immunodeficiency virus type 1 (HIV-1) (29, 34), HIV-2 (2), feline immunodeficiency virus (30), equine infectious anemia virus (24), and more recently simian immunodeficiency virus (SIV) (37). As expected, these lentivirus vectors can achieve gene transfer into growtharrested and differentiated cells ex vivo and in vivo (28).Here we report the construction and characterization of several retroviral vectors based on SIVmac251. The minimal SIV vectors contain less than 20% of the original viral sequences, and recombinant viruses produced by 293T cells were found to carry out very efficient gene transfer into both cycling and growth-arrested cell lines. As a first step toward gene transfer applications, ...

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