Dual-RNAseq Analysis Unravels Virus-Host Interactions of MetSV and Methanosarcina mazei

Methanosarcina spherical virus (MetSV), infecting Methanosarcina species, encodes 22 genes, but their role in the infection process in combination with host genes has remained unknown. To study the infection process in detail, infected and uninfected M. mazei cultures were compared using dual-RNAseq, qRT-PCRs, and transmission electron microscopy (TEM). The transcriptome analysis strongly indicates a combined role of virus and host genes in replication, virus assembly, and lysis. Thereby, 285 host and virus genes were significantly regulated. Within these 285 regulated genes, a network of the viral polymerase, MetSVORF6, MetSVORF5, MetSVORF2, and the host genes encoding NrdD, NrdG, a CDC48 family protein, and a SSB protein with a role in viral replication was postulated. Ultrastructural analysis at 180 min p.i. revealed many infected cells with virus particles randomly scattered throughout the cytoplasm or attached at the cell surface, and membrane fragments indicating cell lysis. Dual-RNAseq and qRT-PCR analyses suggested a multifactorial lysis reaction in potential connection to the regulation of a cysteine proteinase, a pirin-like protein and a HicB-solo protein. Our study's results led to the first preliminary infection model of MetSV infecting M. mazei, summarizing the key infection steps as follows: replication, assembly, and host cell lysis

Rab7b modulates autophagic flux by interacting with Atg4B

Autophagy (macroautophagy) is a highly conserved eukaryotic degradation pathway in which cytosolic components and organelles are sequestered by specialized autophagic membranes and degraded through the lysosomal system. The autophagic pathway maintains basal cellular homeostasis and helps cells adapt during stress; thus, defects in autophagy can cause detrimental effects. It is therefore crucial that autophagy is properly regulated. In this study, we show that the cysteine protease Atg4B, a key enzyme in autophagy that cleaves LC3, is an interactor of the small GTPase Rab7b. Indeed, Atg4B interacts and co‐localizes with Rab7b on vesicles. Depletion of Rab7b increases autophagic flux as indicated by the increased size of autophagic structures as well as the magnitude of macroautophagic sequestration and degradation. Importantly, we demonstrate that Rab7b regulates LC3 processing by modulating Atg4B activity. Taken together, our findings reveal Rab7b as a novel negative regulator of autophagy through its interaction with Atg4B

Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis

In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes

Portuguese history storyboard

This paper intends to present relevant facts about the Portuguese culture and history, so as to enable a better understanding of who the Portuguese are and provide an overall perspective of the course of history in this westernmost part of Europe. Although the choice of historical facts was subjective by nature, it is believed it achieves the aim of presenting information in a critical but blithesome way, with a view to also deconstructing national stereotypes, such as that Portuguese people are always late or are crazy about football. Finally, it focuses on some information about the Portuguese language mainly to serve as a term of comparison with other European languages

THINKING (AND LIVING) EVERYDAY WINE FARM LANDSCAPE AS HORTUS

The study focuses on farmyard landscape quality in wine-growing and producing farms, and is part of a broader research which mainly considers everyday rural landscapes and aims at defining building design criteria for the architectural and landscape quality of agricultural settlements. The research subject of wine production landscapes has been considered since they represent everyday landscapes (European Landscape Convention, art. 2) to which the collective imagery worldwide acknowledges such a high identity value (ELC, art. 5) that they have become emblematic of the culture of several European countries, including Italy. The specific objective of the study is to analyse the main variables that should be considered to design the open spaces of wine-growing and producing farms, with particular reference to Italian small and medium-sized ones. Since they can be found spread over wide areas of the country, they significantly contribute to define its agricultural landscape identity. Given the diversification of Italian wine landscapes, we referred to a study area which proved significant for wine production in Emilia-Romagna in order to contextualize the main issues of landscape design of the wine farm open spaces. The landscape, composition and architectural characters of the various design solutions have been analysed on a representative sample of farms. These analyses have been supported by several on-site surveys, which included interviews to farmers and consumers, surveyed through ad hoc questionnaires and focus groups which also put together professionals employed in specialized firms of the wine-making sector. The analyses allowed to define the main requirements, problems and critical issues related to farmyard design, with reference, on one hand to the various functional aspects of cultivation, harvesting, processing, bottling, and product commercialisation, and on the other hand to aesthetic quality requirements linked to the farm image and marketing strategies. These analyses also allowed to point out some useful references for the quality design of these everyday landscapes. Such indications are discussed in this paper also with reference to a selection of the investigated study cases which proved useful to exemplify the above-mentioned themes

Phthiocerol dimycocerosates promote access to the cytosol and intracellular burden of Mycobacterium tuberculosis in lymphatic endothelial cells

Background: Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs. Results: Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden. Conclusions: PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans

How do barley plants with impaired photosynthetic light acclimation survive under high-light stress?

WHIRLY1 deficient barley plants surviving growth at high irradiance displayed increased non-radiative energy dissipation, enhanced contents of zeaxanthin and the flavonoid lutonarin, but no changes in alpha-tocopherol nor glutathione. Plants are able to acclimate to environmental conditions to optimize their functions. With the exception of obligate shade plants, they can adjust their photosynthetic apparatus and the morphology and anatomy of their leaves to irradiance. Barley (Hordeum vulgare L., cv. Golden Promise) plants with reduced abundance of the protein WHIRLY1 were recently shown to be unable to acclimatise important components of the photosynthetic apparatus to high light. Nevertheless, these plants did not show symptoms of photoinhibition. High-light (HL) grown WHIRLY1 knockdown plants showed clear signs of exposure to excessive irradiance such as a low epoxidation state of the violaxanthin cycle pigments and an early light saturation of electron transport. These responses were underlined by a very large xanthophyll cycle pool size and by an increased number of plastoglobules. Whereas zeaxanthin increased with HL stress, alpha-tocopherol, which is another lipophilic antioxidant, showed no response to excessive light. Also the content of the hydrophilic antioxidant glutathione showed no increase in W1 plants as compared to the wild type, whereas the flavone lutonarin was induced in W1 plants. HPLC analysis of removed epidermal tissue indicated that the largest part of lutonarin was presumably located in the mesophyll. Since lutonarin is a better antioxidant than saponarin, the major flavone present in barley leaves, it is concluded that lutonarin accumulated as a response to oxidative stress. It is also concluded that zeaxanthin and lutonarin may have served as antioxidants in the WHIRLY1 knockdown plants, contributing to their survival in HL despite their restricted HL acclimation

Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo

Effector molecules translocated by the Salmonella pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) critically contribute to the pathogenesis of human Salmonella infection. They facilitate internalization by non-phagocytic enterocytes rendering the intestinal epithelium an entry site for infection. Their function in vivo has remained ill-defined due to the lack of a suitable animal model that allows visualization of intraepithelial Salmonella. Here, we took advantage of our novel neonatal mouse model and analyzed various bacterial mutants and reporter strains as well as gene deficient mice. Our results demonstrate the critical but redundant role of SopE2 and SipA for enterocyte invasion, prerequisite for transcriptional stimulation and mucosal translocation in vivo. In contrast, the generation of a replicative intraepithelial endosomal compartment required the cooperative action of SipA and SopE2 or SipA and SopB but was independent of SopA or host MyD88 signaling. Intraepithelial growth had no critical influence on systemic spread. Our results define the role of SPI1-T3SS effector molecules during enterocyte invasion and intraepithelial proliferation in vivo providing novel insight in the early course of Salmonella infection