Caspase-9 has a nonapoptotic function in Xenopus embryonic primitive blood formation.

Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation.

Environmental Risk Assessment of Recombinant Viral Vector Vaccines against SARS-Cov-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Over the past months, considerable efforts have been put into developing effective and safe drugs and vaccines against SARS-CoV-2. Various platforms are being used for the development of COVID-19 vaccine candidates: recombinant viral vectors, protein-based vaccines, nucleic acid-based vaccines, and inactivated/attenuated virus. Recombinant viral vector vaccine candidates represent a significant part of those vaccine candidates in clinical development, with two already authorised for use in the European Union and one currently under rolling review by the European Medicines Agency (EMA). Since recombinant viral vector vaccine candidates are considered as genetically modified organisms (GMOs), their regulatory oversight includes besides an assessment of their quality, safety and efficacy, also an environmental risk assessment (ERA). The present article highlights the main characteristics of recombinant viral vector vaccine (candidates) against SARS-CoV-2 in the pipeline and discusses their features from an environmental risk point of view.

Opinion: Airtightness for Decontamination by Fumigation of High-Containment Laboratories.

Introduction: While the European legislation states that laboratories of high-containment must be sealable for fumigation, they do not prescribe a minimal value for airtightness. Starting from a previous study in which we measured the airtightness in 4 BSL-3 laboratories with blower-door tests, we discuss the connection between airtightness and a successful decontamination by fumigation. Methods: Biological indicators (BIs) consisting of spores of Geobacillus stearothermophilus on metal disks were laid out in laboratories of different levels of airtightness before performing a fumigation with aerosolized hydrogen peroxide using an automated device, according to the manufacturer's instructions. Results: Incubation of all BI disks placed in the facility with the highest level of airtightness showed complete inactivation of spores. However, in the facility with a lower level of airtightness, not all spores were inactivated. Discussion: Air leaks might be a factor in the outcome of the decontamination of a room by fumigation, as seen in the laboratory with a lower level of airtightness, but other factors associated with the fumigation process might also be critical for a successful decontamination. Conclusion: We argue that a validation of the decontamination procedure, before first use or after important renovations of a laboratory of high-containment, is a more effective endpoint than reaching a predefined level of airtightness.

Identification of drugs that restore primary cilium expression in cancer cells.

The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.

Primary cilium suppression by SREBP1c involves distortion of vesicular trafficking by PLA2G3.

Distortion of primary cilium formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor sterol regulatory element-binding protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently up-regulated in cellular models of SREBP1c-induced cilium repression was phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyzes the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium, as revealed by aberrant transferrin and Rab11 localization, and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells.

Environmental risk assessment of clinical trials involving modified vaccinia virus Ankara (MVA)-based vectors.

The modified vaccinia virus Ankara (MVA) strain, which has been developed as a vaccine against smallpox, is since the nineties widely tested in clinical trials as recombinant vector for vaccination or gene therapy applications. Although MVA is renowned for its safety, several biosafety aspects need to be considered when performing the risk assessment of a recombinant MVA (rMVA). This paper presents the biosafety issues and the main lessons learned from the evaluation of the clinical trials with rMVA performed in Belgium. Factors such as the specific characteristics of the rMVA, the inserted foreign sequences/transgene, its ability for reconversion, recombination and dissemination in the population and the environment are the main points of attention. Measures to prevent or manage identified risks are also discussed.

Aberrant activation of fatty acid synthesis suppresses primary cilium formation and distorts tissue development.

Aberrant activation of fatty acid synthesis is a key feature of many advanced human cancers. Unlike in classical lipogenic tissues, this process has been implicated in membrane production required for rapid cell proliferation. Here, to gain further insight into the consequences of tumor-associated fatty acid synthesis, we have mimicked the lipogenic phenotype of cancer cells in Xenopus embryos by microinjection of RNA encoding the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c). Dramatic morphologic changes were observed that could be linked to alterations in Wnt and Hedgehog signaling, and ultimately to a distortion of the primary cilium. This is a sophisticated microtubular sensory organelle that is expressed on the surface of nearly every cell type and that is lost in many cancers. SREBP1c-induced loss of the primary cilium could be confirmed in mammalian Madin-Darby canine kidney (MDCK) cells and was mediated by changes in the supply of fatty acids. Conversely, inhibition of fatty acid synthesis in highly lipogenic human prostate cancer cells restored the formation of the primary cilium. Lipid-induced ciliary loss was associated with mislocalization of apical proteins, distortion of cell polarization, and aberrant epithelial tissue development as revealed in three-dimensional cultures of MDCK cells and in the developing mouse prostate. These data imply that tumor-associated lipogenesis, in addition to rendering cells more autonomous in terms of lipid supply, disturbs cilium formation and contributes to impaired environmental sensing, aberrant signaling, and distortion of polarized tissue architecture, which are all hallmarks of cancer.

Complete genomic sequence of the goat prion protein gene (PRNP).

Prion protein genetics plays a central role in transmissible spongiform encephalopathies, a disease occurring in human and animals. Here we report a 27-kb genomic sequence containing the goat PRNP gene. It shows, both in structure and content, a remarkable similarity with its sheep ortholog and can serve as a basis for future (comparative) studies with reference to the regulation of PRNP gene expression and the search for genetic tools to prevent/control/eradicate (goat) TSE.