Analysis of Ti- and Pb-based particles in the aqueous environment of Melbourne (Australia) via single particle ICP-MS.

The analysis of natural and anthropogenic nanomaterials (NMs) in the environment is challenging and requires methods capable to identify and characterise structures on the nanoscale regarding particle number concentrations (PNCs), elemental composition, size, and mass distributions. In this study, we employed single particle inductively coupled plasma-mass spectrometry (SP ICP-MS) to investigate the occurrence of NMs in the Melbourne area (Australia) across 63 locations. Poisson statistics were used to discriminate between signals from nanoparticulate matter and ionic background. TiO2-based NMs were frequently detected and corresponding NM signals were calibated with an automated data processing platform. Additionally, a method utilising a larger mass bandpass was developed to screen for particulate high-mass elements. This procedure identified Pb-based NMs in various samples. The effects of different environmental matrices consisting of fresh, brackish, or seawater were mitigated with an aerosol dilution method reducing the introduction of salt into the plasma and avoiding signal drift. Signals from TiO2- and Pb-based NMs were counted, integrated, and subsequently calibrated to determine PNCs as well as mass and size distributions. PNCs, mean sizes, particulate masses, and ionic background levels were compared across different locations and environments.

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

Digital Technologies for Forest Supply Chain Optimization: Existing Solutions and Future Trends.

The role of digital technologies for fostering sustainability and efficiency in forest-based supply chains is well acknowledged and motivated several studies in the scope of precision forestry. Sensor technologies can collect relevant data in forest-based supply chains, comprising all activities from within forests and the production of the woody raw material to its transformation into marketable forest-based products. Advanced planning systems can help to support decisions of the various entities in the supply chain, e.g., forest owners, harvest companies, haulage companies, and forest product processing industry. Such tools can help to deal with the complex interdependencies between different entities, often with opposing objectives and actions-which may increase efficiency of forest-based supply chains. This paper analyzes contemporary literature dealing with digital technologies in forest-based supply chains and summarizes the state-of-the-art digital technologies for real-time data collection on forests, product flows, and forest operations, as well as planning systems and other decision support systems in use by supply chain actors. Higher sustainability and efficiency of forest-based supply chains require a seamless information flow to foster integrated planning of the activities over the supply chain-thereby facilitating seamless data exchange between the supply chain entities and foster new forms of collaboration. Therefore, this paper deals with data exchange and multi-entity collaboration aspects in combination with interoperability challenges related with the integration among multiple process data collection tools and advanced planning systems. Finally, this interdisciplinary review leads to the discussion of relevant guidelines that can guide future research and integration projects in this domain.

Heat stability of foodborne viruses - Findings, methodological challenges and current developments.

Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.

Hyphenation of supercritical fluid chromatography and trapped ion mobility-mass spectrometry for quantitative lipidomics.

BACKGROUND: Lipidomics studies require rapid separations with accurate and reliable quantification results to further elucidate the role of lipids in biological processes and their biological functions. Supercritical fluid chromatography (SFC), in particular, can provide this rapid and high-resolution separation. The combination with trapped ion mobility spectrometry (TIMS) has not yet been applied, although the post-ionization separation method in combination with liquid chromatography or imaging techniques has already proven itself in resolving isomeric and isobaric lipids and preventing false identifications. However, a multidimensional separation method should not only allow confident identification but also provide quantitative results to substantiate studies with absolute concentrations. RESULTS: A SFC method was developed and the hyphenation of SFC and TIMS was further explored towards the separation of different isobaric overlaps. Furthermore, lipid identification was performed using mass spectrometry (MS) and parallel accumulation serial fragmentation (PASEF) MS/MS experiments in addition to retention time and collision cross section (CCS). Quantification was further investigated with short TIMS ramps and performed based on the ion mobility signal of lipids, since TIMS increases the sensitivity by noise filtering. The final method was, as an exemplary study, applied to investigate the function of different ceramide synthases (CerS) in the nematode and model organism Caenorhabditis elegans (C. elegans). Loss of three known CerS hyl-1, hyl-2 and lagr-1 demonstrated different influences on and alterations in the sphingolipidome. SIGNIFICANCE: This method describes for the first time the combination of SFC and TIMS-MS/MS, which enables a fast and sensitive quantification of lipids. The results of the application to C. elegans samples prove the functionality of the method and support research on the metabolism of sphingolipids in nematodes.

High-Voltage Instability of Vinylene Carbonate (VC): Impact of Formed Poly-VC on Interphases and Toxicity.

Full exhaustion in specific energy/energy density of state-of-the-art LiNix Coy Mnz O2 (NCM)-based Li-ion batteries (LIB) is currently limited for reasons of NCM stability by upper cut-off voltages (UCV) below 4.3 V. At higher UCV, structural decomposition triggers electrode crosstalk in the course of enhanced transition metal dissolution and leads to severe specific capacity/energy fade; in the worst case to a sudden death phenomenon (roll-over failure). The additive lithium difluorophosphate (LiDFP) is known to suppress this by scavenging dissolved metals, but at the cost of enhanced toxicity due to the formation of organofluorophosphates (OFPs). Addition of film-forming electrolyte additives like vinylene carbonate (VC) can intrinsically decrease OFP formation in thermally aged LiDFP-containing electrolytes, though the benefit of this dual-additive approach can be questioned at higher UCVs. In this work, VC is shown to decrease the formation of potentially toxic OFPs within the electrolyte during cycling at conventional UCVs but triggers OFP formation at higher UCVs. The electrolyte contains soluble VC-polymerization products. These products are formed at the cathode during VC oxidation (and are found within the cathode electrolyte interphase (CEI), suggesting an OFP electrode crosstalk of VC decomposition species, as the OFP-precursor molecules are shown to be formed at the anode.

Platial mobility: expanding place and mobility in GIS via platio-temporal representations and the mobilities paradigm.

While platial representations are being developed for sedentary entities, a parallel and useful endeavor would be to consider time in so-called "platio-temporal" representations that would also expand notions of mobility in GIScience, that are solely dependent on Euclidean space and time. Besides enhancing such aspects of place and mobility via spatio-temporal, we also include human aspects of these representations via considerations of the sociological notions of mobility via the mobilities paradigm that can systematically introduce representation of both platial information along with mobilities associated with 'moving places.' We condense these aspects into 'platial mobility,' a novel conceptual framework, as an integration in GIScience and the mobilities paradigm in sociology, that denotes movement of places in our platio-temporal and sociology-based representations. As illustrative cases for further study using platial mobility as a framework, we explore its benefits and methodological aspects toward developing better understanding for disaster management, disaster risk reduction and pandemics. We then discuss some of the illustrative use cases to clarify the concept of platial mobility and its application prospects in the areas of disaster management, disaster risk reduction and pandemics. These use cases, which include flood events and the ongoing COVID-19 pandemic, have led to displaced and restricted communities having to change practices and places, which would be particularly amenable to the conceptual framework developed in our work.

The Translated Amino Acid Sequence of an Insertion in the Hepatitis E Virus Strain 47832c Genome, But Not the RNA Sequence, Is Essential for Efficient Cell Culture Replication.

The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography - mass spectrometry.

Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Reverse genetics approaches for hepatitis E virus and related viruses.

The hepatitis E virus (HEV) is the causative agent of acute and chronic hepatitis in humans. Related viruses have been found in several animal species. Reverse genetics systems (RGSs), which enable the generation of infectious virus from cloned cDNA by transfection of cultured cells or intrahepatic injection into laboratory animals, have been developed for HEV genotypes 1, 3, 4, 5 and 7 as well as for avian HEV and rat HEV. However, low virus recovery rates and slow replication in cell cultures are observed for most of the HEV types. Nevertheless, the RGSs enabled the site-directed mutagenesis of single nucleotides, deletion of genome fragments, insertion of sequence tags and a marker gene as well as the generation of chimeric viruses.

Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c.

The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.

Benefits of intraoperative digital subtraction angiography in primary arteriovenous fistula creation.

Digital subtraction angiography (DSA) is an ideal procedure for improving vascular surgery results, not yet in use for arteriovenous fistula (AVF) creation. In a prospective study, the practicability and benefits of intraoperative completion DSA (CDSA) for this purpose were investigated. When the arteriovenous anastomosis was completed and clinically judged as running, DSA via percutaneous or open-site puncture was conducted. Findings and consequences were prospectively documented. From July 2005 to September 2006 a primary AVF was created in 54 patients. Fifty of these could be included in the study (68, 39-91 yrs). In 44 a forearm fistula, and in six patients an elbow fistula was newly created. In two cases we were unable to insert a cannula into the A. brachialis (study escape). In the remaining 48 cases, CDSA visualized afferent artery, anastomosis and draining vein. No complications occurred associated with CDSA; 3.5, 2-12 ml, contrast agent were required per patient. In 13 cases, CDSA revealed problems with the AVF that could be corrected immediately: the vein was freed from restraining soft tissue (n=4), a stealing venous branch was ligated (n=6), transluminal thrombectomy was conducted (n=1) or anastomosis was newly created (n=3). AVF could be successfully created proven by ongoing dialysis in 30 cases, and in eight cases as demonstrated by clinical and sonographic evaluations, resulting in a primary 1-yr patency rate of 79.2%. CDSA in AVF surgery is practicable and reveals conditions that can be corrected immediately, thus improving surgical outcome. Long since standard in other areas of vascular surgery, CDSA should become a routine procedure in AVF creation.

Bone and soft tissue palatal morphology and potential anchorage sides in cleft palate patients.

AIM: The aim of this study was to evaluate palatal vertical bone thickness and density in relation to soft tissue on the hard palate for better selection of adequate bone regions for the insertion of orthodontic mini-implants (MIs) in cleft palate patients. MATERIALS AND METHODS: Cone beam computed tomography scans (CBCT) were obtained from 60 patients (mean age range 9-12). The study population included patients with isolate right side cleft palate formation (n = 20; 6 females; 14 males), left side cleft palate formation (n = 20; 9 females; 11 males) and without cleft formation as control group (n = 20; 15 females; 5 males). Bone and soft tissue measurements were performed vertical at a 90° angle to the bone surface, on previously defined measurement points (n = 88) on the hard palate. Bone density was measured on ten vertical layers in caudo-cranial direction. RESULTS: In non-cleft patient the highest bone thickness was in the anterior palate and decreased significantly in posterior direction. In patients with right and left cleft palate, the highest vertical bone level could be observed at the palatal premaxillary border opposite to the cleft side. Patients in the control group showed a significantly lower vertical soft tissue thickness than patients with palatal cleft formation. The evaluation of bone density showed no significant differences in all three groups. CONCLUSION: The results suggest that the favorable region for orthodontic MI placement is in the similar anatomical region compared to non-cleft patients, but differs from one side in each group. In unilateral cleft palate patients, the highest bone level was found on the anterior palate side opposite to the cleft side, indicating the most effective region for MIs placement.

Mitoferrin-1 is required for brain energy metabolism and hippocampus-dependent memory.

Disturbed iron (Fe) ion homeostasis and mitochondrial dysfunction have been implicated in neurodegeneration. Both processes are related, because central Fe ion consuming biogenetic pathways take place in mitochondria and affect their oxidative energy metabolism. Iron is imported into mitochondria by the two homologous Fe ion importers mitoferrin-1 and mitoferrin-2. To elucidate more specifically the role of mitochondrial Fe ions for brain energy metabolism and for proper neuronal function, we generated mice with a neuron-specific knockout of mitoferrin-1 (Slc25a37-/- or mfrn-1-/-) and compared them with corresponding control littermates (mfrn-1flox/flox). Mice lacking neuronal mfrn-1 exhibited no obvious anatomical or behavioral abnormalities as neonates, young or adult animals. However, they exhibited a moderate decrease in brain mitochondrial O2-consumption with complex-I substrates of the electron transport chain (p < 0.05), indicating a moderate suppression of electron transport. While these mice did not exhibit altered basal fear levels, inquisitiveness or motor skills in specific neurobiological test batteries, they clearly exhibited decreased spatial learning skills and missing establishment of stable spatial memory in Morris water maze, as compared to floxed controls (p < 0.05). We thus conclude that mitochondrial Fe ion supply is an important player in neuronal energy metabolism and proper brain function and that the carrier mitoferrin-1 cannot be completely replaced by mitoferrin-2 or other as yet unknown Fe ion carriers.

Frequent AKT1E17K mutations in skull base meningiomas are associated with mTOR and ERK1/2 activation and reduced time to tumor recurrence.

BACKGROUND: Skull base meningiomas are considered to be difficult for surgical treatment. We wondered whether genetic alterations recently identified in benign non-NF2-mutated World Health Organization (WHO) grade I meningiomas are related to clinical features of skull base meningiomas and whether druggable signaling pathways are activated. METHODS: We analyzed 93 skull base meningiomas (82 WHO grade I, 11 WHO grade II) for mutations of hot spots or the most relevant exons of AKT1, KLF4/TRAF7, SMO, PI3K, and the TERT promoter. RESULTS: The AKT1E17K mutation was present in 31% of patients and was related to meningothelial histology. AKT1E17K had a negative effect on the time to tumor recurrence. Analyses of activated signaling proteins revealed among AKT1E17K tumors a significantly higher rate of phospho-mammalian target of rapamycin (mTOR) and phospho-p70S6K+ tumors. AKT1E17K tumors with immunoexpression of phospho-extracellular signal-regulated kinase 1 or 2 (ERK1/2) were characterized by significantly shorter time to tumor recurrence compared with AKT1wt tumors expressing phospho-ERK1/2 (P = .046). KLF4 mutations (K409Q) were present in 11.8% of cases, with significant association to the secretory/transitional subtype (P < .001). The presence of the KLF4 K409Q mutation was associated with favorable outcome. One phosphatidylinositol-3 kinase (PI3K) mutation but no SMO or TERT promoter mutation was found. CONCLUSIONS: AKT1E17K mutation is frequent in skull base meningiomas, results in activation of the mTOR and ERK1/2 signaling pathways, and has negative impact on tumor recurrence. Patients with skull base meningiomas with AKT1E17K mutation might benefit from additional treatment targeting the mTOR pathway. Generally, the PI3K-Akt-mTOR axis might be a potential target for kinase inhibitors in these tumors.

Receptor tyrosine kinase inhibition by regorafenib/sorafenib inhibits growth and invasion of meningioma cells.

Systemic chemotherapeutic treatment for unresectable and/or aggressive meningiomas is still unsatisfying. PDGF receptor (PDGFR)-mediated activation of mitogenic signalling has been shown to be active in meningiomas. Therefore, we evaluate in vitro and in vivo the effects of inhibiting PDGFR using the clinically well-characterised tyrosine kinase inhibitors sorafenib or regorafenib in meningioma models. IOMM-Lee meningioma cells were used to assess cytotoxic effects, inhibition of proliferation, induction of apoptosis, as well as inhibition of migration and motility by sorafenib and regorafenib. Using an orthotopic mouse xenograft model, growth inhibition as monitored by magnetic resonance imaging, and overall survival of sorafenib- or regorafenib-treated mice compared with control animals was determined. Treatment of malignant IOMM-Lee cells resulted in significantly reduced cell survival and induction of apoptosis following regorafenib and sorafenib treatment. Western blots showed that both drugs target phosphorylation of p44/42 ERK via downregulation of the PDGFR. Both drugs additionally showed significant inhibition of cell motility and invasion. In vivo, mice with orthotopic meningioma xenografts showed a reduced volume (n.s.) of signal enhancement in MRI (mainly tumour) following sorafenib and regorafenib treatment. This was translated in a significantly increased overall survival time (p ≤ 0.05) for regorafenib-treated mice. Analyses of in vivo-grown tumours demonstrated again reduced PDGFR expression and expression/phosphorylation of p44/42. Sorafenib and regorafenib show antitumour activity in vitro and in vivo by targeting PDGFR and p44/42 ERK signalling.