Critical Reactions in Fluorobenzoic Acid Degradation by Pseudomonas sp. B13

3-Chlorobenzoate-grown cells of Pseudomonas sp. B13 readily cometabolized monofluorobenzoates. A catabolic pathway for the isomeric fluorobenzoates is proposed on the basis of key metabolites isolated. Only 4-fluorobenzoate was utilized and totally degraded after a short period of adaptation. The isoenzymes for total degradation of chlorocatechols, being found during growth with 3-chlorobenzoate or 4-chlorophenol, were not induced in the presence of fluorobenzoates. Correspondingly, only the ordinary enzymes of the benzoate pathway were detected in 4-fluorobenzoate-grown cells. Ring cleavage of 3-fluorocatechol was recognized as a critical step in 3-fluorobenzoate degradation. 2-Fluoro-cis,cis-muconic acid was identified as a dead-end metabolite from 2- and 3-fluorobenzoate catabolism. During 2-fluorobenzoate cometabolism, fluoride is eliminated by the initial dioxygenation.     

Inflammation and metabolic dysfunction: links to cardiovascular diseases

Abdominal obesity is a major risk factor for cardiovascular disease, and recent studies highlight a key role of adipose tissue dysfunction, inflammation, and aberrant adipokine release in this process. An increased demand for lipid storage results in both hyperplasia and hypertrophy, finally leading to chronic inflammation, hypoxia, and a phenotypic change of the cellular components of adipose tissue, collectively leading to a substantially altered secretory output of adipose tissue. In this review we have assessed the adipo-vascular axis, and an overview of adipokines associated with cardiovascular disease is provided. This resulted in a first list of more than 30 adipokines. A deeper analysis only considered adipokines that have been reported to impact on inflammation and NF-κB activation in the vasculature. Out of these, the most prominent link to cardiovascular disease was found for leptin, TNF-α, adipocyte fatty acid-binding protein, interleukins, and several novel adipokines such as lipocalin-2 and pigment epithelium-derived factor. Future work will need to address the potential role of these molecules as biomarkers and/or drug targets.     

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.     

Nucleic Acid Tools for Invasive Fungal Disease Diagnosis

Current Fungal Infection Reports - This review has incorporated the knowledge and experience of the leads of each of the laboratory working parties of the fungal PCR initiative in order to provide...     

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.     

Scission of COPI and COPII Vesicles Is Independent of GTP Hydrolysis

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP‐binding proteins. Some reports describe that the scission of COP‐coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI‐ and COPII‐coated vesicles utilizing semi‐intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP‐PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.     

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

Microsatellite markers reveal clear geographic structuring among threatened noble crayfish (Astacus astacus) populations in Northern and Central Europe

Noble crayfish (Astacus astacus L.), the most highly valued freshwater crayfish in Europe, is threatened due to a long-term population decline caused mainly by the spread of crayfish plague. Reintroduction of the noble crayfish into restored waters is a common practice but the geographic and genetic origin of stocking material has rarely been considered, partially because previous genetic studies have been hampered by lack of nuclear gene markers with known inheritance. This study represents the first large scale population genetic survey of the noble crayfish (633 adults from 18 locations) based on 10 newly developed microsatellite markers. We focused primarily on the Baltic Sea area (Estonia, Finland and Sweden) where the largest proportion of the remaining populations exists. To allow comparisons, samples from the Black Sea catchment (the Danube drainage) were also included. Two highly differentiated population groups were identified corresponding to the Baltic Sea and the Black Sea catchments, respectively. The Baltic Sea catchment populations had significantly lower genetic variation and private allele numbers than the Black Sea catchment populations. Within the Baltic Sea area, a clear genetic structure was revealed with population samples corresponding well to their geographic origin, suggesting little impact of long-distance translocations. The clear genetic structure strongly suggests that the choice of stocking material for re-introductions and supplemental releases needs to be based on empirical genetic knowledge.