HUN-REN Szegedi Biológiai Kutatóközpont

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV‐mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd‐sourcing, drawing on the unique EV expertise of academia‐based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.

As the result of intensive research and breeding efforts over the last 20 years, the yield potential and yield quality of cereals have been greatly improved. Nowadays, yield safety has gained more importance because of the forecasted climatic changes. Drought and high temperature are especially considered as key stress factors with high potential impact on crop yield. Yield safety can only be improved if future breeding attempts will be based on the valuable new knowledge acquired on the processes determining plant development and its responses to stress. Plant stress responses are very complex. Interactions between plant structure, function and the environment need to be investigated at various phases of plant development at the organismal, cellular as well as molecular levels in order to obtain a full picture. The results achieved so far in this field indicate that various plant organs, in a definite hierarchy and in interaction with each other, are involved in determining crop yield under stress. Here we attempt to summarize the currently available information on cereal reproduction under drought and heat stress and to give an outlook towards potential strategies to improve yield safety in cereals.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Circadian clocks are believed to confer an advantage to plants, but the nature of that advantage has been unknown. We show that a substantial photosynthetic advantage is conferred by correct matching of the circadian clock period with that of the external light-dark cycle. In wild type and in long- and short-circadian period mutants of Arabidopsis thaliana, plants with a clock period matched to the environment contain more chlorophyll, fix more carbon, grow faster, and survive better than plants with circadian periods differing from their environment. This explains why plants gain advantage from circadian control.

Another host factor for SARS-CoV-2 Virus-host interactions determine cellular entry and spreading in tissues. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the earlier SARS-CoV use angiotensin-converting enzyme 2 (ACE2) as a receptor; however, their tissue tropism differs, raising the possibility that additional host factors are involved. The spike protein of SARS-CoV-2 contains a cleavage site for the protease furin that is absent from SARS-CoV (see the Perspective by Kielian). Cantuti-Castelvetri et al. now show that neuropilin-1 (NRP1), which is known to bind furin-cleaved substrates, potentiates SARS-CoV-2 infectivity. NRP1 is abundantly expressed in the respiratory and olfactory epithelium, with highest expression in endothelial and epithelial cells. Daly et al. found that the furin-cleaved S1 fragment of the spike protein binds directly to cell surface NRP1 and blocking this interaction with a small-molecule inhibitor or monoclonal antibodies reduced viral infection in cell culture. Understanding the role of NRP1 in SARS-CoV-2 infection may suggest potential targets for future antiviral therapeutics. Science , this issue p. 856 , p. 861 ; see also p. 765

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.

To optimize their growth and survival, plants perceive and respond to ultraviolet-B (UV-B) radiation. However, neither the molecular identity of the UV-B photoreceptor nor the photoperception mechanism is known. Here we show that dimers of the UVR8 protein perceive UV-B, probably by a tryptophan-based mechanism. Absorption of UV-B induces instant monomerization of the photoreceptor and interaction with COP1, the central regulator of light signaling. Thereby this signaling cascade controlled by UVR8 mediates UV-B photomorphogenic responses securing plant acclimation and thus promotes survival in sunlight.

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Francis Martin and colleagues report genome sequences for 18 species of mycorrhizal fungi and a phylogenomic analysis including 32 other fungal genomes. The study identifies cell wall-degradation genes lost in all true ectomycorrhizal species and, using gene expression data, finds candidate genes for the establishment of symbiosis. To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall–degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7–38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This "blood-brain barrier" function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood-brain barrier models with a focus on their validation regarding a set of well-established blood-brain barrier characteristics. As an ideal cell culture model of the blood-brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

Abstract Single-cell technologies have transformed our understanding of human tissues. Yet, studies typically capture only a limited number of donors and disagree on cell type definitions. Integrating many single-cell datasets can address these limitations of individual studies and capture the variability present in the population. Here we present the integrated Human Lung Cell Atlas (HLCA), combining 49 datasets of the human respiratory system into a single atlas spanning over 2.4 million cells from 486 individuals. The HLCA presents a consensus cell type re-annotation with matching marker genes, including annotations of rare and previously undescribed cell types. Leveraging the number and diversity of individuals in the HLCA, we identify gene modules that are associated with demographic covariates such as age, sex and body mass index, as well as gene modules changing expression along the proximal-to-distal axis of the bronchial tree. Mapping new data to the HLCA enables rapid data annotation and interpretation. Using the HLCA as a reference for the study of disease, we identify shared cell states across multiple lung diseases, including SPP1 + profibrotic monocyte-derived macrophages in COVID-19, pulmonary fibrosis and lung carcinoma. Overall, the HLCA serves as an example for the development and use of large-scale, cross-dataset organ atlases within the Human Cell Atlas.

With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.

Plants under oxidative stress suffer from damages that have been interpreted as unavoidable consequences of injuries inflicted upon plants by toxic levels of reactive oxygen species (ROS). However, this paradigm needs to be modified. Inactivation of a single gene, EXECUTER1, is sufficient to abrogate stress responses of Arabidopsis thaliana caused by the release of singlet oxygen: External conditions under which these stress responses are observed and the amounts of ROS that accumulate in plants exposed to these environmental conditions do not directly cause damages. Instead, seedling lethality and growth inhibition of mature plants result from genetic programs that are activated after the release of singlet oxygen has been perceived by the plant.

A method is introduced to build microscopic light driven rotors that are created and manipulated in laser tweezers. Laser light-induced two-photon polymerization of light curing resins is applied to generate effective rotating particles several microns in size. The dynamics of rotation are evaluated. Mechanical devices consisting of multiple moving parts driven by these rotors are produced. It is shown that the combination of the techniques of microfabrication by two-photon polymerization by light and the light-induced trapping and rotation offers a powerful tool with which to build complex mechanical machines of micrometer size.

Patients with type 2 diabetes have reduced gene expression of heat shock protein (HSP) 72, which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signaling proteins such as c-jun amino terminal kinase (JNK), inhibitor of kappaB kinase, and tumor necrosis factor-alpha, can induce insulin resistance, but HSP 72 can block the induction of these molecules in vitro. Accordingly, we examined whether activation of HSP72 can protect against the development of insulin resistance. First, we show that obese, insulin resistant humans have reduced HSP72 protein expression and increased JNK phosphorylation in skeletal muscle. We next used heat shock therapy, transgenic overexpression, and pharmacologic means to overexpress HSP72 either specifically in skeletal muscle or globally in mice. Herein, we show that regardless of the means used to achieve an elevation in HSP72 protein, protection against diet- or obesity-induced hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance was observed. This protection was tightly associated with the prevention of JNK phosphorylation. These findings identify an essential role for HSP72 in blocking inflammation and preventing insulin resistance in the context of genetic obesity or high-fat feeding.

Dendritic cells are potent antigen-presenting cells that initiate and amplify immune responses. To determine whether dendritic cells participate in inflammatory reactions in amyotrophic lateral sclerosis (ALS), we examined mRNA expression of dendritic cell surface markers in individual sporadic ALS (sALS), familial ALS (fALS), and nonneurological disease control (NNDC) spinal cord tissues using semiquantitative and real-time reverse transcription polymerase chain reaction (RT-PCR). Immature (DEC205, CD1a) and activated/mature (CD83, CD40) dendritic cell transcripts were significantly elevated in ALS tissues. The presence of immature and activated/mature dendritic cells (CD1a(+) and CD83(+)) was confirmed immunohistochemically in ALS ventral horn and corticospinal tracts. Monocytic/macrophage/microglial transcripts (CD14, CD18, SR-A, CD68) were increased in ALS spinal cord, and activated CD68(+) cells were demonstrated in close proximity to motor neurons. mRNA expressions of the chemokine MCP-1, which attracts monocytes and myeloid dendritic cells, and of the cytokine macrophage-colony stimulating factor (M-CSF) were increased in ALS tissues. The MCP-1 protein was expressed in glia in ALS but not in control tissues and was increased in the CSF of ALS patients. Those patients who progressed most rapidly expressed significantly more dendritic transcripts than patients who progressed more slowly. These results support the involvement of immune/inflammatory responses in amplifying motor neuron degeneration in ALS.

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.

Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.