Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches

Single Cell analysis using AtomicForce Microscopy (AFM)

Replication of biological cells for the purpose of imaging and analysis under electron and scanning probe microscopy has facilitated the opportunity to study and examine some molecular processes and structures of living cells in a manner that were not possible before. The difficulties faced in direct cellular analysis when using and operating Atomic Force Microscopy (AFM) in situ for morphological studies of biological cells have led to the development of a novel method for biological cell studies based on nanoimprint lithography. The realization of the full potential of high resolution AFM imaging has revealed some very important biological events such as exocytosis and endocytosis. In this work, a soft lithography Bioimprint replication technique, which involved simple fabrication steps, was used to form a hard replica of the cell employing a newly developed biocompatible polymer that has fast curing time at room temperature essential for this process. The structure and topography of the rat muscle cell and the endometrial (Ishikawa) cancer cell were investigated in this study. Cells were cultured and incubated in accordance with standard biological culturing procedures and protocols approved by the Human Ethics Committee, University of Otago. An impression of the cell profile was created by applying a layer of the polymer onto the cells attached to a substrate and rapidly cured under UV-light. Fast UV radiation helps to lock cellular processes within seconds after exposure and replicas of the cancer cells exhibit ultra-cellular structures and features down to nanometer scale. Elimination of the AFM tip damping effects due to probing of the soft biological tissue allows imaging with unprecedented resolution. Highxx resolution AFM imagery provides the opportunity to examine the structure and topography of the cells closely so that any abnormalities can be identified. Craters that resemble granules and features down to 100 nm were observed. These represent steps on a transitional series of sequential structures that indicate either an endocytotic or exocytotic processes, which were evident on the replicas. These events, together with exocytosis, play a very significant part in the tumorigenesis of these cancer cells. By forming cell replica impressions, not only have they the potential to understand biological cell conditions, but may also benefit in synthesizing three dimensional (3-D) scaffolds for natural growth of biological cells and providing an improvement over standard cell growth conditions. Further examinations by observing the characteristic behaviour of the plasma membrane when the cells were induced by certain compound such as cobalt chloride (CoCl2) under control and stimulated conditions have brought in the opportunity to examine the effect of this stimulant in inducing apoptosis in many different kinds of cells. Numbers of pores formed on the cells membrane were found to increase significantly after the cells where induced with CoCl2 that correlated well with the level of vascular endothelial growth factor (VEGF) receptors expression, which contributed to tumour growth. This indicates CoCl2 has exaggerated the expression of the VEGF growth factor. Investigations were also done to the cells using functionalized nanoparticles as bio-markers to establish the connection between exocytosis with nanopores found on the membrane surfaces of the cells. These microbeads were found attached to sites surrounding the nucleus of the cell and higher numbers of visible beads would confirm that there was an up-regulation of the VEGF expression in cells induced by CoCl2. All these can contribute to expanding the knowledge about exocytosis and fundamental physiology of cells, and also assist in understanding diseases especially cancer

Entwicklungs- und Designmethoden für hochintegrale Leichtbauteile aus Faser-Kunststoff-Verbundmaterial

Aus der Einleitung: "Faser-Kunststoff-Verbund (FKV)-Werkstoffe finden einen immer breiteren Einsatz in allen Bereichen der Industrie, wie zum Beispiel in der Luft- und Raumfahrt, im Automotive-Bereich, im Maschinenbau und bei Sportgeräten. Dabei entstehen besondere Herausforderungen für Entwickler, da Eigenschaften und Verfahren im Zusammenhang dieser Werkstoffe sich deutlich von denen der herkömmlich verwendeten Metalle oder unverstärkten Kunststoffe unterscheiden. Technische Fasern werden in verschiedenen Verarbeitungsformen und in Kombination mit vielfältigen Matrixsystemen angewendet. Ein Großteil der Fasern wird heutzutage in Form von multiaxialen Geweben oder Gelegen verarbeitet. Bei diesen Halbzeugen sind die Fasern in mehreren Lagen unterschiedlicher Ausrichtung übereinandergelegt. Metalle können damit sehr einfach durch leichtere Faser- Kunststoffverbunde ersetzt werden. Diese Technologien versuchen weitestgehend isotrope Bauteileigenschaften aus den eigentlich anisotropen Materialeigenschaften zu erzielen. Dies reizt jedoch das Potential der Werkstoffe nicht aus.

Key factors in simulating the equatorial Atlantic zonal sea surface temperature gradient in a coupled general circulation model

Causes of the coupled model bias in simulating the zonal sea surface temperature (SST) gradient in the equatorial Atlantic are examined in three versions of the same coupled general circulation model (CGCM) differing only in the cumulus convection scheme. One version of the CGCM successfully simulates the mean zonal SST gradient of the equatorial Atlantic, in contrast to the failure of the Coupled Model Intercomparison Project phase 3 models. The present analysis shows that key factors to be successful are high skills in simulating the meridional location of the Intertropical Convergence Zone, the precipitation over northern South America, and the southerly winds along the west coast of Africa associated with the West African monsoon in boreal spring. Model biases in the Pacific contribute to the weaker precipitation over northern South America. Uncoupled experiments with the atmospheric component further confirm the importance of remote influences on the development of the equatorial Atlantic bias. Key Points: The zonal SST gradient of the equatorial Atlantic is well simulated in a CGCM; Key factors for the realistic simulation of the Atlantic SST are presented; Remote forcing from the Pacific may contribute to the Atlantic SST bia

Challenges in quantifying changes in the global water cycle

Human influences have likely already impacted the large-scale water cycle but natural variability and observational uncertainty are substantial. It is essential to maintain and improve observational capabilities to better characterize changes. Understanding observed changes to the global water cycle is key to predicting future climate changes and their impacts. While many datasets document crucial variables such as precipitation, ocean salinity, runoff, and humidity, most are uncertain for determining long-term changes. In situ networks provide long time-series over land but are sparse in many regions, particularly the tropics. Satellite and reanalysis datasets provide global coverage, but their long-term stability is lacking. However, comparisons of changes among related variables can give insights into the robustness of observed changes. For example, ocean salinity, interpreted with an understanding of ocean processes, can help cross-validate precipitation. Observational evidence for human influences on the water cycle is emerging, but uncertainties resulting from internal variability and observational errors are too large to determine whether the observed and simulated changes are consistent. Improvements to the in situ and satellite observing networks that monitor the changing water cycle are required, yet continued data coverage is threatened by funding reductions. Uncertainty both in the role of anthropogenic aerosols, and due to large climate variability presently limits confidence in attribution of observed changes

The tropical Atlantic observing system

The tropical Atlantic is home to multiple coupled climate variations covering a wide range of timescales and impacting societally relevant phenomena such as continental rainfall, Atlantic hurricane activity, oceanic biological productivity, and atmospheric circulation in the equatorial Pacific. The tropical Atlantic also connects the southern and northern branches of the Atlantic meridional overturning circulation and receives freshwater input from some of the world’s largest rivers. To address these diverse, unique, and interconnected research challenges, a rich network of ocean observations has developed, building on the backbone of the Prediction and Research Moored Array in the Tropical Atlantic (PIRATA). This network has evolved naturally over time and out of necessity in order to address the most important outstanding scientific questions and to improve predictions of tropical Atlantic severe weather and global climate variability and change. The tropical Atlantic observing system is motivated by goals to understand and better predict phenomena such as tropical Atlantic interannual to decadal variability and climate change; multidecadal variability and its links to the meridional overturning circulation; air-sea fluxes of CO2 and their implications for the fate of anthropogenic CO2; the Amazon River plume and its interactions with biogeochemistry, vertical mixing, and hurricanes; the highly productive eastern boundary and equatorial upwelling systems; and oceanic oxygen minimum zones, their impacts on biogeochemical cycles and marine ecosystems, and their feedbacks to climate. Past success of the tropical Atlantic observing system is the result of an international commitment to sustained observations and scientific cooperation, a willingness to evolve with changing research and monitoring needs, and a desire to share data openly with the scientific community and operational centers. The observing system must continue to evolve in order to meet an expanding set of research priorities and operational challenges. This paper discusses the tropical Atlantic observing system, including emerging scientific questions that demand sustained ocean observations, the potential for further integration of the observing system, and the requirements for sustaining and enhancing the tropical Atlantic observing system

Nonproductive exposure of PBMCs to SARS‐CoV ‐2 induces cell‐intrinsic innate immune responses

Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Equatorial Atlantic Ocean dynamics in a coupled ocean–atmosphere model simulation

The ocean temperatures and zonal currents at the equatorial Atlantic simulated by an improved version of the Brazilian earth system model (BESM), with changes in the cloud cover scheme and optical properties of the atmospheric component, are analyzed and compared to those obtained from a previous version of BESM and also from other seven selected CMIP5 models. It is shown that this updated version of BESM, despite some persistent biases, more accurately represents the surface temperature variation at the Equator and the equatorial thermocline east–west slope. These improvements are associated to a more realistic seasonal cycle achieved for the Atlantic equatorial undercurrent, as well as sea surface temperatures and zonal wind stress. The better simulation of the equatorial undercurrent is, in its turn, credited to a more realistic representation of the surface wind position and strength at the tropical Atlantic by the coupled model. With many of the systematic errors noticed in the previous version of the model alleviated, this version of BESM can be considered as a useful tool for modelers involved in Atlantic variability studies