Biosafety and biosecurity challenges during the COVID-19 pandemic and beyond.

As the world continues to battle the SARS-CoV-2 pandemic, it is a stark reminder of the devastation biological threats can cause. In an unprecedented way the global community saw a massive surge in the demand for diagnostic capacities, which had a substantial impact on biosafety and biosecurity. Laboratories had to cope with a surge in laboratory testing capacity, while resources and training possibilities were limited. In addition, the pandemic highlighted the impact biological threats can have, thereby giving rise to new dialogue about biosecurity and new biological threats. This paper aims to highlight some of the most pressing issues regarding biosafety and biosecurity observed during the COVID-19 pandemic with special focus on low and lower middle-income countries. The authors provide lessons learned, tools and recommendations to improve future biosafety and biosecurity and increase preparedness for the next global health crisis.

SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD.

SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld-/- and Spata2-/- mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld-/-Spata2-/- fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld-/-Spata2-/- macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld-/- or Spata2-/- cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld-/-Spata2-/- cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling.

Molecular Characterisation and Phylogeny of Tula Virus in Kazakhstan.

Orthohantaviruses are zoonotic pathogens that play a significant role in public health. These viruses can cause haemorrhagic fever with renal syndrome in Eurasia. In the Republic of Kazakhstan, the first human cases were registered in the year 2000 in the West Kazakhstan region. Small mammals can be reservoirs of orthohantaviruses. Previous studies showed orthohantavirus antigens in wild-living small mammals in four districts of West Kazakhstan. Clinical studies suggested that there might be further regions with human orthohantavirus infections in Kazakhstan, but genetic data of orthohantaviruses in natural foci are limited. The aim of this study was to investigate small mammals for the presence of orthohantaviruses by molecular biological methods and to provide a phylogenetic characterization of the circulating strains in Kazakhstan. Small mammals were trapped at 19 sites in West Kazakhstan, four in Almaty region and at seven sites around Almaty city during all seasons of 2018 and 2019. Lung tissues of small mammals were homogenized and RNA was extracted. Orthohantavirus RT-PCR assays were applied for detection of partial S and L segment sequences. Results were compared to published fragments. In total, 621 small mammals from 11 species were analysed. Among the collected small mammals, 2.4% tested positive for orthohantavirus RNA, one sample from West Kazakhstan and 14 samples from Almaty region. None of the rodents caught in Almaty city were infected. Sequencing parts of the small (S) and large (L) segments specified Tula virus (TULV) in these two regions. Our data show that geographical distribution of TULV is more extended as previously thought. The detected sequences were found to be split in two distinct genetic clusters of TULV in West Kazakhstan and Almaty region. TULV was detected in the common vole (Microtus arvalis) and for the first time in two individuals of the forest dormouse (Dryomys nitedula), interpreted as a spill-over infection in Kazakhstan.

First Indications of Omsk Haemorrhagic Fever Virus beyond Russia.

Omsk haemorrhagic fever virus (OHFV) is the agent leading to Omsk haemorrhagic fever (OHF), a viral disease currently only known in Western Siberia in Russia. The symptoms include fever, headache, nausea, muscle pain, cough and haemorrhages. The transmission cycle of OHFV is complex. Tick bites or contact with infected small mammals are the main source of infection. The Republic of Kazakhstan is adjacent to the endemic areas of OHFV in Russia and febrile diseases with haemorrhages occur throughout the country-often with unclear aetiology. In this study, we examined human cerebrospinal fluid samples of patients with suspected meningitis or meningoencephalitis with unknown origins for the presence of OHFV RNA. Further, reservoir hosts such as rodents and ticks from four Kazakhstan regions were screened for OHFV RNA to clarify if this virus could be the causative agent for many undiagnosed cases of febrile diseases in humans in Kazakhstan. Out of 130 cerebrospinal fluid samples, two patients (1.53%) originating from Almaty city were positive for OHFV RNA. Screening of tick samples revealed positive pools from different areas in the Akmola region. Of the caught rodents, 1.1% out of 621 were positive for OHFV at four trapping areas from the West Kazakhstan region. In this paper, we present a broad investigation of the spread of OHFV in Kazakhstan in human cerebrospinal fluid samples, rodents and ticks. Our study shows for the first time that OHFV can not only be found in the area of Western Siberia in Russia, but can also be detected up to 1.600 km away in the Almaty region in patients and natural foci.

Tick-borne encephalitis virus and West-Nile fever virus as causes of serous meningitis of unknown origin in Kazakhstan.

Flaviviruses are a family of viruses that cause many diseases in humans. Their similarity in the antigenic structure causes a cross-reaction, which complicates the precise diagnostic of disease causing agents. Tick-borne encephalitis virus (TBEV), a member of the flavivirus family, is the cause of tick-borne encephalitis (TBE). Worldwide the awareness of this disease is raising, however, in many countries such as the Republic of Kazakhstan (KZ) there is a lack of serological investigation of flaviviruses in humans. In our study, we focused on two TBE endemic regions of KZ (East Kazakhstan Oblast (EKO) and Almaty (AO)) and a region where TBE cases were registered only since 2010 (Akmola Oblast (AkO)). In KZ, up to 400 cases of serous meningitis of unknown origin were registered annually in the period from 2017 to 2019. Our goals were to calculate the prevalence of antibodies against TBEV in patients with suspected meningitis. We collected 179 sera and 130 cerebrospinal fluid (CSF) samples from patients and included a questionnaire with focus on socio-demographical factors and observed tick bites. The human samples were tested with TBEV and West-Nile fever virus (WNFV) IgM and IgG ELISA, by immunofluorescence assay using a flavivirus biochip, and TBEV-specific real-time RT-PCR. We found TBEV and WNFV antibodies in 31 samples by serological and molecular techniques. Seven serum samples out of 31 showed TBEV-specific antibodies, and three serum pairs had WNFV antibodies. Correlating the serological results with the information gained from the questionnaires it becomes apparent that the number of tick bites is a significant factor for a TBEV infection. This result has an impact on diagnostic in KZ and physicians should be aware that both flaviviruses play a role for serous meningitis of unknown origin in KZ.

Advances in and the Applicability of Machine Learning-Based Screening and Early Detection Approaches for Cancer: A Primer.

Despite the efforts of the past decades, cancer is still among the key drivers of global mortality. To increase the detection rates, screening programs and other efforts to improve early detection were initiated to cover the populations at a particular risk for developing a specific malignant condition. These diagnostic approaches have, so far, mostly relied on conventional diagnostic methods and have made little use of the vast amounts of clinical and diagnostic data that are routinely being collected along the diagnostic pathway. Practitioners have lacked the tools to handle this ever-increasing flood of data. Only recently, the clinical field has opened up more for the opportunities that come with the systematic utilisation of high-dimensional computational data analysis. We aim to introduce the reader to the theoretical background of machine learning (ML) and elaborate on the established and potential use cases of ML algorithms in screening and early detection. Furthermore, we assess and comment on the relevant challenges and misconceptions of the applicability of ML-based diagnostic approaches. Lastly, we emphasise the need for a clear regulatory framework to responsibly introduce ML-based diagnostics in clinical practice and routine care.

Eight Years of Collaboration on Biosafety and Biosecurity Issues Between Kazakhstan and Germany as Part of the German Biosecurity Programme and the G7 Global Partnership Against the Spread of Weapons and Materials of Mass Destruction.

In 2013, the German Federal Foreign Office launched the German Biosecurity Programme with the aim to minimise risks associated with biological substances and pathogens. In this context, the German-Kazakh Network for Biosafety and Biosecurity was established in 2013 and constitutes a successful collaboration between Kazakh and German biomedical organisations, under the co-management of the Bundeswehr Institute of Microbiology (IMB), and the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH. Ever since then, a network of scientists, stake holders and policymakers has been established, aiming to work on highly pathogenic, potential biological warfare agents with the focus on biosafety and biosecurity, surveillance, detection and diagnostics, networking and awareness raising of these agents in Kazakhstan. Over the past 8 years, the project members trained four PhD candidates, organised over 30 workshops and trainings with more than 250 participants and conducted more than 5,000 PCR assays and 5,000 serological analyses for surveillance. A great success was the description of new endemic areas for Orthohantaviruses, the mixture of two Crimean-Congo haemorrhagic fever virus genetic clusters, new foci and genetic information on tick-borne encephalitis virus and rickettsiae in Kazakh oblasts. The latter even led to the description of two new genogroups. Furthermore, joint contributions to international conferences were made. In this report, we summarise the evolution of the German-Kazakh Network for Biosafety and Biosecurity and critically reflect on the strengths and possible weaknesses. We were able to establish a viable network of biosafety and biosecurity shareholders and to accomplish the aims of the German Biosecurity Programme to lower biosecurity risks by increased awareness, improved detection and diagnostic methods and surveillance. Further, we reflect on forthcoming aspects to lead this interstate endeavour into a sustainable future.

Loss of PKD1/polycystin-1 impairs lysosomal activity in a CAPN (calpain)-dependent manner.

Mutations in the PKD1 gene result in autosomal dominant polycystic kidney disease (ADPKD), the most common monogenetic cause of end-stage renal disease (ESRD) in humans. Previous reports suggested that PKD1, together with PKD2/polycystin-2, may function as a receptor-cation channel complex at cilia and on intracellular membranes and participate in various signaling pathways to regulate cell survival, proliferation and macroautophagy/autophagy. However, the exact molecular function of PKD1 and PKD2 has remained enigmatic. Here we used Pkd1-deficient mouse inner medullary collecting duct cells (mIMCD3) genetically deleted for Pkd1, and tubular epithelial cells isolated from nephrons of doxycycline-inducible conditional pkd1fl/fl;Pax8rtTA;TetOCre+ knockout mice to show that the lack of Pkd1 caused diminished lysosomal acidification, LAMP degradation and reduced CTSB/cathepsin B processing and activity. This led to an impairment of autophagosomal-lysosomal fusion, a lower delivery of ubiquitinated cargo from multivesicular bodies (MVB)/exosomes to lysosomes and an enhanced secretion of unprocessed CTSB into the extracellular space. The TFEB-dependent lysosomal biogenesis pathway was however unaffected. Pkd1-deficient cells exhibited increased activity of the calcium-dependent CAPN (calpain) proteases, probably due to a higher calcium influx. Consistent with this notion CAPN inhibitors restored lysosomal function, CTSB processing/activity and autophagosomal-lysosomal fusion, and blocked CTSB secretion and LAMP degradation in pkd1 knockout cells. Our data reveal for the first time a lysosomal function of PKD1 which keeps CAPN activity in check and ensures lysosomal integrity and a correct autophagic flux.Abbreviations: acCal: acetyl-calpastatin peptide; ADPKD: autosomal dominant polycystic kidney disease; CI-1: calpain inhibitor-1; CQ: chloroquine; Dox: doxycycline; EV: extracellular vesicles; EXO: exosomes; LAMP1/2: lysosomal-associated membrane protein 1/2; LGALS1/GAL1/galectin-1: lectin, galactose binding, soluble 1; LMP: lysosomal membrane permeabilization; mIMCD3: mouse inner medullary collecting duct cells; MV: microvesicles; MVB: multivesicular bodies; PAX8: paired box 8; PKD1/polycystin-1: polycystin 1, transient receptor potential channel interacting; PKD2/polycystin-2: polycystin 2, transient receptor potential cation channel; Tet: tetracycline; TFEB: transcription factor EB; VFM: vesicle-free medium; WT: wild-type.

Automated spheroid generation, drug application and efficacy screening using a deep learning classification: a feasibility study.

The last two decades saw the establishment of three-dimensional (3D) cell cultures as an acknowledged tool to investigate cell behaviour in a tissue-like environment. Cells growing in spheroids differentiate and develop different characteristics in comparison to their two-dimensionally grown counterparts and are hence seen to exhibit a more in vivo-like phenotype. However, generating, treating and analysing spheroids in high quantities remains labour intensive and therefore limits its applicability in drugs and compound research. Here we present a fully automated pipetting robot that is able to (a) seed hanging drops from single cell suspensions, (b) treat the spheroids formed in these hanging drops with drugs and (c) analyse the viability of the spheroids by an image-based deep learning based convolutional neuronal network (CNN). The model is trained to classify between 'unaffected', 'mildly affected' and 'affected' spheroids after drug exposure. All corresponding spheroids are initially analysed by viability flow cytometry analysis to build a labelled training set for the CNN to subsequently reduce the number of misclassifications. Hence, this approach allows to efficiently examine the efficacy of drug combinatorics or new compounds in 3D cell culture. Additionally, it may provide a valuable instrument to screen for new and individualized systemic therapeutic strategies in second and third line treatment of solid malignancies using patient derived primary cells.

Cells grown in three-dimensional spheroids mirror in vivo metabolic response of epithelial cells.

Metabolism in cells adapts quickly to changes in nutrient availability and cellular differentiation status, including growth conditions in cell culture settings. The last decade saw a vast increase in three-dimensional (3D) cell culture techniques, engendering spheroids and organoids. These methods were established to improve comparability to in vivo situations, differentiation processes and growth modalities. How far spheroids mimic in vivo metabolism, however, remains enigmatic. Here, to our knowledge, we compare for the first time metabolic fingerprints between cells grown as a single layer or as spheroids with freshly isolated in situ tissue. While conventionally grown cells express elevated levels of glycolysis intermediates, amino acids and lipids, these levels were significantly lower in spheroids and freshly isolated primary tissues. Furthermore, spheroids differentiate and start to produce metabolites typical for their tissue of origin. 3D grown cells bear many metabolic similarities to the original tissue, recommending animal testing to be replaced by 3D culture techniques.

TNFα sensitizes hepatocytes to FasL-induced apoptosis by NFκB-mediated Fas upregulation.

Although it is well established that TNFα contributes to hepatitis, liver failure and associated hepatocarcinogenesis via the regulation of inflammation, its pro-apoptotic role in the liver has remained enigmatic. On its own, TNFα is unable to trigger apoptosis. However, when combined with the transcriptional inhibitor GaLN, it can cause hepatocyte apoptosis and liver failure in mice. Moreover, along with others, we have shown that TNFα is capable of sensitizing cells to FasL- or drug-induced cell death via c-Jun N-terminal kinase (JNK) activation and phosphorylation/activation of the BH3-only protein Bim. In this context, TNFα could exacerbate hepatocyte cell death during simultaneous inflammatory and T-cell-mediated immune responses in the liver. Here we show that TNFα sensitizes primary hepatocytes, established hepatocyte cell lines and mouse embryo fibroblasts to FasL-induced apoptosis by the transcriptional induction and higher surface expression of Fas via the NFκB pathway. Genetic deletion, diminished expression or dominant-negative inhibition of the NFκB subunit p65 resulted in lower Fas expression and inhibited TNFα-induced Fas upregulation and sensitization to FasL-induced cell death. By hydrodynamic injection of p65 shRNA into the tail vein of mice, we confirm that Fas upregulation by TNFα is also NFκB-mediated in the liver. In conclusion, TNFα sensitization of FasL-induced apoptosis in the liver proceeds via two parallel signaling pathways, activation of JNK and Bim phosphorylation and NFκB-mediated Fas upregulation.

Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Role of apoptosis in the development of autosomal dominant polycystic kidney disease (ADPKD).

Autosomal dominant polycystic kidney disease (ADPKD) is a widespread genetic disorder in the Western world and is characterized by cystogenesis that often leads to end-stage renal disease (ESRD). Mutations in the pkd1 gene, encoding for polycystin-1 (PC1) and its interaction partner pkd2, encoding for polycystin-2 (PC2), are the main drivers of this disease. PC1 and PC2 form a multiprotein membrane complex at cilia sites of the plasma membrane and at intracellular membranes. This complex mediates calcium influx and stimulates various signaling pathways regulating cell survival, proliferation and differentiation. The molecular consequences of pkd1 and pkd2 mutations are still a matter of debate. In particular, the ways in which the cysts are initially formed and progress throughout the disease are unknown. The mechanisms proposed to play a role include enhanced cell proliferation, increased apoptotic cell death and diminished autophagy. In this review, we summarize our current understanding about the contribution of apoptosis to cystogenesis and ADPKD. We present the animal models and the tools and methods that have been created to analyze this process. We also critically review the data that are in favor or against the involvement of apoptosis in disease generation. We argue that apoptosis is probably not the sole driver of cystogenesis but that a cooperative action of cell death, compensatory cell proliferation and perturbed autophagy gradually establish the disease. Finally, we propose novel strategies for uncovering the mode of action of PC1 and PC2 and suggest means by which their dysfunction or loss of expression lead to cystogenesis and ADPKD development.

Possible pitfalls investigating cell death responses in genetically engineered mouse models and derived cell lines.

Genetically engineered mouse models are frequently used to identify pathophysiological consequences of deregulated cell death. Targeting pro-apoptotic or anti-apoptotic proteins of the extrinsic or intrinsic apoptotic signalling cascade is state of the art since more than two decades. Such animal models have been increasingly made use of over the past years to study loss- or gain-of-function consequences of one or more components of the molecular machinery leading to cell death. These studies have helped to separate redundant from non-redundant functions of apoptosis-related proteins in normal physiology and sometimes unravelled unexpected phenotypes. However, correct interpretation of data derived from knockout mice or derived cells and cell lines is often flawed by the comparison of cells originating from different inbred or mixed genetic backgrounds. Here we want to highlight some basic problems associated with genetic background-based modulation of cell death sensitivity and describe some methods that we use to investigate cell death responses in hematopoietic and non-hematopoietic cells. Thereby, we show that hematopoietic cells derived from wild type mice on a C57BL/6:129/SvJ recombinant mixed genetic background are significantly more resistant to spontaneous cell death or DNA-damage induced apoptosis in vitro than cells derived from inbred C57BL/6 mice. Furthermore, we show as an example that C57BL/6 mice are more susceptible to γ-irradiation induced cell death after whole body irradiation in vivo and subsequent T cell lymphomagenesis.