Mutational analysis of c-KIT and PDGFRA receptors in gastrointestinal stromal tumours

Introduction: The pathogenesis of gastrointestinal stromal tumors (GISTs) is generally associated with activating mutations of the proto-oncogene tyrosine-protein kinase Kit (c-KIT). However, about 15% of GISTs do not harbour c-KIT mutations. It is estimated that 5% of these GISTs have mutations in the platelet-derived growth factor receptor α (PDGFRA). Accurate diagnosis of GIST has become very important since the availability of targeted therapy with tyrosine kinase inhibitors, such as imatinib mesylate. The routine workup for GIST diagnosis includes immunohistochemistry for CD117 (c-KIT polyclonal antibody), as it is estimated that 95% of GIST cases show positive immunoreactivity. However, it can be observed that the routinely used immunohistochemical analysis does not provide complete sensitivity for GIST diagnosis, as there are nearly 5% of GISTs that are negative for c-KIT immunohistochemistry. Mutational analysis for c-KIT and PDGFRA can confirm the diagnosis of GIST, particularly in CD117-negative suspect GIST. Moreover, specific mutations have a prognostic and/or a predictive value for response to therapy.peer-reviewe

Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish

Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain–sDMA interactions, piRNAs and piRNA targets

Orthogonal Separation Techniques for the Characterization of the Yeast Nuclear Proteome

The presence of the nucleus is the distinguishing feature of eukaryotic cells, separating the genome from the cytoplasm. Key cellular events, including transcription, DNA replication, RNA-processing and ribosome biogenesis all take place in the nucleus. All of these processes can be regulated through controlled and bidirectional translocation of proteins across the nuclear envelope, making the nucleus a highly dynamic organelle. In this study, we present four orthogonal multidimensional separation techniques for the comprehensive characterization of the yeast nuclear proteome. By combining methods on the peptide level (SCX chromatography, isoelectric focusing) and protein level (SDS-PAGE, phosphocellulose chromatography) coupled with mass spectrometry, we identified 1889 proteins from highly purified nuclei, of which 1032 were previously annotated as nuclear proteins. In particular, the most successful setup was the use of phosphocellulose P11 chromatography in combination with SDS-PAGE and reversed phase chromatography. Phosphocellulose P11 chromatography has been classically used for the purification of functional protein complexes involved in transcription regulation. Here, by its coupling with LC-MS, this method resulted in approximately 1.5 times more protein identifications than the other three combined, thereby contributing significantly to the coverage of nuclear proteins. In addition, the use of this technique resulted in the enrichment of DNA binding proteins and proved to be a valuable tool for the simultaneous analysis of multiple protein complexes. The enrichment for specific nuclear complexes has resulted in high protein sequence coverage, which will be particularly useful for the detailed characterization of subunits

Orthogonal Separation Techniques for the Characterization of the Yeast Nuclear Proteome

The presence of the nucleus is the distinguishing feature of eukaryotic cells, separating the genome from the cytoplasm. Key cellular events, including transcription, DNA replication, RNA-processing and ribosome biogenesis all take place in the nucleus. All of these processes can be regulated through controlled and bidirectional translocation of proteins across the nuclear envelope, making the nucleus a highly dynamic organelle. In this study, we present four orthogonal multidimensional separation techniques for the comprehensive characterization of the yeast nuclear proteome. By combining methods on the peptide level (SCX chromatography, isoelectric focusing) and protein level (SDS-PAGE, phosphocellulose chromatography) coupled with mass spectrometry, we identified 1889 proteins from highly purified nuclei, of which 1032 were previously annotated as nuclear proteins. In particular, the most successful setup was the use of phosphocellulose P11 chromatography in combination with SDS-PAGE and reversed phase chromatography. Phosphocellulose P11 chromatography has been classically used for the purification of functional protein complexes involved in transcription regulation. Here, by its coupling with LC-MS, this method resulted in approximately 1.5 times more protein identifications than the other three combined, thereby contributing significantly to the coverage of nuclear proteins. In addition, the use of this technique resulted in the enrichment of DNA binding proteins and prove

Assessment of a DNA vaccine encoding Burkholderia pseudomallei bacterioferritin

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in Southeast Asia and Northern Australia. The bacteria cause infection via subcutaneous or inhaled routes, resulting in either acute lethal sepsis or chronic and eventually fatal disease. Currently no licensed vaccine is available to provide protection against this pathogen. Intracellular enzymatic proteins of other bacterial species, such as the iron storage protein bacterioferritin, have been shown to be potent inducers of the immune response. In this study, a DNA vaccine encoding the B. pseudomallei bacterioferritin protein was constructed. The DNA vaccine was then used to immunise mice and analyse subsequent immune responses and protective capability following live challenge with B. pseudomallei. There was a substantial increase in anti-bacterioferritin IgG titers following immunisation, however the cellular response and survival following challenge was limited, suggesting that the vaccine may need to be used in conjunction with adjuvant such as CpG or in a multicomponent vaccine in order to increase protective capabilities

Tdrd1 acts as a molecular scaffold for Piwi proteins and piRNA targets in zebrafish

Piwi proteins function in an RNAi-like pathway that silences transposons. Piwi-associated RNAs, also known as piRNAs, act as a guide to identify Piwi targets. The tudor domain-containing protein Tdrd1 has been linked to this pathway but its function has thus far remained unclear. We show that zebrafish Tdrd1 is required for efficient Piwi-pathway activity and proper nuage formation. Furthermore, we find that Tdrd1 binds both zebrafish Piwi proteins, Ziwi and Zili, and reveals sequence specificity in the interaction between Tdrd1 tudor domains and symmetrically dimethylated arginines (sDMAs) in Zili. Finally, we show that Tdrd1 complexes contain piRNAs and RNA molecules that are longer than piRNAs. We name these longer transcripts Tdrd1-associated transcripts (TATs). TATs likely represent cleaved Piwi pathway targets and may serve as piRNA biogenesis intermediates. Altogether, our data suggest that Tdrd1 acts as a molecular scaffold for Piwi proteins, bound through specific tudor domain–sDMA interactions, piRNAs and piRNA targets

Digitalizing and Upgrading Severe Acute Respiratory Infections Surveillance in Malta: System Development

BackgroundIn late 2020, the European Centre for Disease Prevention and Control and Epiconcept started implementing a surveillance system for severe acute respiratory infections (SARI) across Europe. ObjectiveWe sought to describe the process of digitizing and upgrading SARI surveillance in Malta, an island country with a centralized health system, during the COVID-19 pandemic from February to November 2021. We described the characteristics of people included in the surveillance system and compared different SARI case definitions, including their advantages and disadvantages. This study also discusses the process, output, and future for SARI and other public health surveillance opportunities. MethodsMalta has one main public hospital where, on admission, patient data are entered into electronic records as free text. Symptoms and comorbidities are manually extracted from these records, whereas other data are collected from registers. Collected data are formatted to produce weekly and monthly reports to inform public health actions. From October 2020 to February 2021, we established an analogue incidence-based system for SARI surveillance. From February 2021 onward, we mapped key stakeholders and digitized most surveillance processes. ResultsBy November 30, 2021, 903 SARI cases were reported, with 380 (42.1%) positive for SARS-CoV-2. Of all SARI hospitalizations, 69 (7.6%) were admitted to the intensive care unit, 769 (85.2%) were discharged, 27 (3%) are still being treated, and 107 (11.8%) died. Among the 107 patients who died, 96 (89.7%) had more than one underlying condition, the most common of which were hypertension (n=57, 53.3%) and chronic heart disease (n=49, 45.8%). ConclusionsThe implementation of enhanced SARI surveillance in Malta was completed by the end of May 2021, allowing the monitoring of SARI incidence and patient characteristics. A future shift to register-based surveillance should improve SARI detection through automated processes

Host responses to melioidosis and tuberculosis are both dominated by interferon-mediated signaling.

Melioidosis (Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Northeast Thailand and northern Australia. B. pseudomallei is a soil saprophyte endemic to Southeast Asia and northern Australia. The clinical presentation of melioidosis may mimic tuberculosis (both cause chronic suppurative lesions unresponsive to conventional antibiotics and both commonly affect the lungs). The two diseases have overlapping risk profiles (e.g., diabetes, corticosteroid use), and both B. pseudomallei and Mycobacterium tuberculosis are intracellular pathogens. There are however important differences: the majority of melioidosis cases are acute, not chronic, and present with severe sepsis and a mortality rate that approaches 50% despite appropriate antimicrobial therapy. By contrast, tuberculosis is characteristically a chronic illness with mortality <2% with appropriate antimicrobial chemotherapy. We examined the gene expression profiles of total peripheral leukocytes in two cohorts of patients, one with acute melioidosis (30 patients and 30 controls) and another with tuberculosis (20 patients and 24 controls). Interferon-mediated responses dominate the host response to both infections, and both type 1 and type 2 interferon responses are important. An 86-gene signature previously thought to be specific for tuberculosis is also found in melioidosis. We conclude that the host responses to melioidosis and to tuberculosis are similar: both are dominated by interferon-signalling pathways and this similarity means gene expression signatures from whole blood do not distinguish between these two diseases