Remote sensing of bush encroachment on commercial cattle farms in semi-arid rangelands in Namibia

Bush encroachment is one of the most extensive changes in land cover in Namibia and an urgent problem for cattle farming, rapidly reducing the productivity of the rangeland. Despite the severity of these consequences, a complete and accurate assessment of bush encroached areas is still missing at large. This study aims at assessing bush encroachment on commercial cattle farms in central Namibia by employing remote sensing methods to distinguish between areas covered by bush and open rangeland. Herein we use different classification techniques and vegetation indices to characterize the nature of vegetation cover. Our analysis shows that results are sensitive to specific classifications of indices. As an accuracy assessment could not be run on these results we could not analyze which classification approximates real bush encroachment best. Hence, this study highlights the need for further analysis. Ground truth data, in the form of field mappings, high resolution aerial photographs or local expert knowledge are needed to gain further insights and produce reliable results.remote sensing, semi-arid rangelands, cattle farming, bush encroachment

Nucleocapsid-specific T cell responses associate with control of SARS-CoV-2 in the upper airways before seroconversion

Despite intensive research since the emergence of SARS-CoV-2, it has remained unclear precisely which components of the early immune response protect against the development of severe COVID-19. Here, we perform a comprehensive immunogenetic and virologic analysis of nasopharyngeal and peripheral blood samples obtained during the acute phase of infection with SARS-CoV-2. We find that soluble and transcriptional markers of systemic inflammation peak during the first week after symptom onset and correlate directly with upper airways viral loads (UA-VLs), whereas the contemporaneous frequencies of circulating viral nucleocapsid (NC)-specific CD4+ and CD8+ T cells correlate inversely with various inflammatory markers and UA-VLs. In addition, we show that high frequencies of activated CD4+ and CD8+ T cells are present in acutely infected nasopharyngeal tissue, many of which express genes encoding various effector molecules, such as cytotoxic proteins and IFN-γ. The presence of IFNG mRNA-expressing CD4+ and CD8+ T cells in the infected epithelium is further linked with common patterns of gene expression among virus-susceptible target cells and better local control of SARS-CoV-2. Collectively, these results identify an immune correlate of protection against SARS-CoV-2, which could inform the development of more effective vaccines to combat the acute and chronic illnesses attributable to COVID-19

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Functional Characterization of Periosteum-derived Cell Subpopulations by Means of Magnetic Cell Separations

Tissue Engineering Anwendungen im Bereich der Mund-, Kiefer- und Gesichtschirurgie bieten eine geeignete Alternativtherapie zu der Standardtherapie der autologen Knochentransplantation für die Regeneration von Knochendefekten. Hierzu ist neben geeigneten Trägermaterialien sowie Differenzierungsfaktoren eine mesenchymale Stammzellquelle für die Besiedelung der Konstrukte nötig. Mesenchymale Stammzellen (MSCs) finden sich im menschlichen adulten Körper in nahezu allen Gewebearten. Als eine der gängigsten Quellen hat sich das Knochenmark etabliert. Ebenso befinden sich osteogene Vorläuferzellen in der Knochenhaut (Periost), die in vivo den Aufbau von neuem Knochen verantwortet. Durch die leichtere Zugänglichkeit sowie die Funktionalität bietet das Kieferperiost im Vergleich zum Knochenmark eine geeignetere Quelle für MSCs im Anwendungsbereich der Mund-, Kiefer- und Gesichtschirurgie an. Da sich die unterschiedlichen Zellschichten der humanen Knochenhaut nur sehr schwer trennen lassen, ist es eine Herausforderung, für die osteogenen Vorläuferzellen aus der Kambiumschicht geeignete Marker zu identifizieren, um nur diese aus dem Gesamtverband zuverlässig isolieren zu können. Verschiedene spezifische Oberflächenmarker für MSCs wurden bereits beschrieben, darunter CD90, CD73, CD105, CD271, Stro-1 und MSCA-1. Letzterer definiert eine Subpopulation aus der Kieferknochenhaut mit höherem osteogenen Potenzial. Da die gängige und gut etablierte Standardmethode der magnetischen Zellseparation (MACS) sowie die durchflusszytometrische Zellsortierung meist recht hohe Mortalitätsraten mit sich ziehen, besteht der Bedarf einer schonenderen Alternativmethode für die Zellseparation. Dafür muss jedoch der genaue, detaillierte Vergleich verschiedener Separationsmethoden gezogen werden. Mit Hilfe von magnetisch markierten MSCA-1 Antikörper wurden in der vorliegenden Arbeit zwei magnetische Separationsmethoden verglichen und daraufhin untersucht eine möglichst reine MSC-haltige Subpopulation aus der heterogenen Periostzellkultur zu isolieren. Neben der etablierten MACS-Methode, die auf einem Säulenprinzip beruht wurde eine weitere magnetische Zellseparationsmethode (EasySep) untersucht, bei der weniger Scherkräfte entstehen. Diese beiden Separationsmethoden wurden in der vorliegenden Arbeit zum ersten Mal vergleichend auf ihre Zellausbeuten, Mortalitätsraten, Zellreinheit und das Beibehalten der Differenzierbarkeit untersucht. Beide untersuchten Methoden unterschieden sich signifikant bezüglich der resultierenden Überlebensraten: dabei zeigte die EasySep Methode signifikant niedrigere Mortalitätsraten (20,32%; MACS: 41,98%). Mithilfe von durchflusszytometrischen Analysen konnte jedoch gezeigt werden, dass die MACS separierten Fraktionen die höhere Reinheit aufwiesen: während der Anteil an MSCA-1+ Zellen in der Positivfraktion bei etwa 45% lag, betrug der Anteil MSCA-1+ Zellen in der entsprechenden Ea-sySep separierten Fraktion nur 19%. Überraschenderweise zeigte die EasySep separierte Negativfraktion einen höheren Anteil an MSCA-1+ Zellen. Weiterhin konnte nur für die MACS Methode eine höhere Mineralisationskapazität der MSCA-1+ Frak-tion im Vergleich zur Negativkontrolle nachgewiesen werden, wohingegen die Fraktionen von EasySep keine Unterschiede in Bezug auf das osteogene Differenzierungspotential zeigten. Die Untersuchung der mitochondrialen Aktivität ergab signifikante Unterschiede nur in den MACS separierten Fraktionen nach osteogener Stimulation. Im Laufe der in vitro Passagierung zeigten die Zellfraktionen beider Methoden eine Abnahme der Mineralisations- und Proliferationskapazität sowie eine ansteigende Seneszenz. Betrachtet man diese Ergebnisse und bringt sie in Verbindung mit den untersuchten Genen und Proteinen, so kommt man zu dem Schluss, dass die MACS Methode eine für Tissue Engineering Anwendungen besser geeignete MSCA-1+ Subpopulation aus der heterogenen Periostzellkultur isoliert. Die für die Zellen schonendere, aber weniger effiziente EasySep Methode brachte keine überzeugenden Resultate ein.Tissue engineering applications in the field of oral and maxillofacial surgery provide a suitable alternative to the standard therapy of autologous bone transplantation for the regeneration of bone defects. Besides the need of suitable scaffolds and differentiation factors an adequate source of mesenchymal stem cells is required for seeding of the developed constructs. In the human body mesenchymal stem cells (MSCs) are found throughout almost all kinds of tissue. Bone marrow is up to date the best-established source of MSCs. Osteogenic progenitors are also found within the periosteum being responsible for the in vivo new bone formation. Due to the better acces-sibility and the high functionality, periosteum tissue represents a suitable source of MSCs for bone regeneration issues in the oral and maxillofacial surgery compared to bone marrow. Due to the fact that the different layers of jaw periosteum are hardly to separate, the identification of suitable markers for the reliable isolation of osteoprogenitors from the whole tissue poses a challenge. Different surface markers of MSCs have already been described, such as CD90, CD73, CD105, CD271, Stro-1 and MSCA-1. The latter one defines a subpopulation of the jaw periosteum eliciting a higher osteogenic potential. The well-established standard method for magnetic cell separation (MACS) shows high mortality rates. Therefore the establishment of a gentler alternative for cell separation and a detailed and exact comparison of different methods are required. In the present work two magnetic separation methods using specific MSCA-1 antibodies were compared regarding the isolation of a pure MSC-containing subpopulation from the heterogeneous population of the jaw periosteum. Besides the use of the well-established MACS separation method based on a column set-up, a second magnetic cell separation method (EasySep) providing a set-up with lower shearforces was performed. Both separation methods have been compared regarding cell yields, mortality rates, cell purities and the ability to differentiate into osteogenic tissue. The analyzed methods showed significant differences regarding the cell viability: the EasySep approach showed significant lower mortality rates (20.32% in comparison to 41.98% by MACS). Using flow cytometric analysis we provided evidence that MACS isolated cell fractions showed the higher purities: in the MACS separated MSCA-1positive cell fraction we detected 45% MSCA-1+ cells, whereas the EasySep fraction contained only 19% MSCA-1+ cells. Surprisingly, the negative fraction of EasySep showed the higher amount of MSCA-1+ cells. The MACS separated MSCA-1+ fraction revealed a significant higher mineralization capacity compared to the negative fraction, whereas the EasySep separated fractions showed no differences regarding the osteogenic potential. We detected significant differences considering the mitochondrial activity designating the proliferative capacities of cells only in MACS separated fractions after osteogenic stimulation. During in vitro passaging of the isolated cell fractions a decrease of the mineralization and proliferation capacities of cells and an increase in cell senescence was observed independent from the separation method. Considering these results and those analyzed by gene and protein expression, we can conclude that the MACS method provides the more suitable approach to isolate a pure MSCA-1+ subpopulation from the heterogeneous jaw periosteum for tissue engineering applications in the oral and maxillofacial surgery. The gentler but less efficient EasySep method yielded no convincing results

Isolation of osteoprogenitors from human jaw periosteal cells: a comparison of two magnetic separation methods.

Human jaw periosteum tissue contains osteoprogenitors that have potential for tissue engineering applications in oral and maxillofacial surgeries. To isolate osteoprogenitor cells from heterogeneous cell populations, we used the specific mesenchymal stem cell antigen-1 (MSCA-1) antibody and compared two magnetic separation methods. We analyzed the obtained MSCA-1(+) and MSCA-1(-) fractions in terms of purity, yield of positive/negative cells and proliferative and mineralization potentials. The analysis of cell viability after separation revealed that the EasySep method yielded higher viability rates, whereas the flow cytometry results showed a higher purity for the MACS-separated cell fractions. The mineralization capacity of the osteogenic induced MSCA-1(+) cells compared with the MSCA-1(-) controls using MACS was 5-fold higher, whereas the same comparison after EasySep showed no significant differences between both fractions. By analyzing cell proliferation, we detected a significant difference between the proliferative potential of the osteogenic cells versus untreated cells after the MACS and EasySep separations. The differentiated cells after MACS separation adjusted their proliferative capacity, whereas the EasySep-separated cells failed to do so. The protein expression analysis showed small differences between the two separation methods. Our findings suggest that MACS is a more suitable separation method to isolate osteoprogenitors from the entire jaw periosteal cell population

Cell proliferation behaviour during JPC osteogenesis.

<p>The proliferation rates (optical densities) of the MACS- (A, C) and EasySep-separated (B, D) MSCA-1^+/− cells were analyzed at days 5, 10 and 20 of the osteogenic differentiation (n = 4 for each separation method) compared with the untreated controls. The MACS-separated MSCA-1^−/+ cells at day 20 showed significant differences (p<0,044) between the undifferentiated cells and the osteogenic induced ones for the MSCA-1⁻ and MSCA-1⁺ cells. In contrast, the EasySep-separated MSCA-1^−/+ cells proliferated almost identically in osteogenic stimulated and untreated controls. The significant decrease in proliferation of the MACS-separated compared to the EasySep-separated positive fraction at day 20 is shown in part E of the figure (p<0.05).</p

Survival rates after MACS (green column) and EasySep (blue column) separation.

<p>For the MACS separation, about 40% and for the EasySep separation about 20% of the initially applied cells were lost. The difference between the survival/mortality rates obtained by both separation methods (n = 16 for each separation method) was significant (p<0.0001).</p

Cell purity of MACS (A, C) and EasySep (B, D) separated cell fractions as determined by flow cytometric analysis.

<p>After the magnetic separations, the cells were labeled with the anti-MSCA-1 supernatants and the PE-labeled secondary antibodies and MSCA-1 expression was analyzed by FACS. In the upper panel of the figure, representative flow cytometric histograms of MSCA-1^−/+ are illustrated. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047176#pone-0047176-g002" target="_blank">Fig. 2C and 2D</a> show the results of the quantification of the MSCA-1⁺ cells within the MACS/EasySep separated fractions (n = 4). MSCA-1 expression was significantly different (p<0,021) between the MACS- but not EasySep-separated MSCA-1⁻ (11.15±3.17) and MSCA-1⁺ (44.26±5.24) cell fractions.</p

Quantification of the mineralization capacity of the MACS- (bright and dark green columns) and EasySep- (bright and dark blue columns) separated cell fractions.

<p>After Alizarin Red staining and subsequent dissolving of the dye from the monolayers, calcium phosphate precipitates were photometrically quantitated (n = 13 for each separation method). The increase in amounts of calcium-phosphate precipitates compared with the untreated controls (induction index, x-fold) is illustrated. A 5-fold higher amount of precipitates was detected in the MACS-separated MSCA-1⁺ compared with the MSCA-1⁻ cells (p<0.05) (A). In contrast, EasySep-separated cell fractions did not significantly differ concerning their mineralization capacity (B). The direct comparison of the positive fractions separated by both methods showed no significant differences, as shown in part C of the figure.</p