Novel mechanism of plasmid-DNA transfer mediated by heterologous cell fusion in syntrophic coculture of Clostridium organisms

The evolution of bacteria is driven by random genetic mutations and horizontal gene transfer (HGT) of genetic material from other bacteria. HGT can occur via transformation, transduction, and conjugation. Here, we present a potential new mechanism of HGT which occurs in a syntrophic Clostridium coculture. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, the two organisms undergo heterologous cell fusion, which includes fusion of the peptidoglycan cell walls and membranes. Heterologous cell fusion facilitated a large-scale exchange of cytoplasmic protein and RNA between the two organisms, leading to the formation of hybrid bacterial cells containing cytoplasmic material of the two parent organisms. Here we present new evidence that cell fusion events also facilitate the exchange of plasmid DNA between the two organisms of the syntrophic coculture. Through the use of a selective subculturing process, we successfully isolated wild-type C. acetobutylicum clones which have acquired a portion of the plasmid DNA, containing the antibiotic resistance marker, from a recombinant strain of C. ljungdahlii. Fusion events led to formation of persistent aberrant hybrid cells with distinct morphogenetic characteristics. Furthermore, our data support the concept of a novel, interspecies, mechanism of acquiring antibiotic resistance. Since neither organism contains any known conjugation machinery or mechanism, these findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.

Autophagy: A Friend or Foe in Allergic Asthma?

Autophagy is a major self-degradative process through which cytoplasmic material, including damaged organelles and proteins, are delivered and degraded in the lysosome. Autophagy represents a dynamic recycling system that produces new building blocks and energy, essential for cellular renovation, physiology, and homeostasis. Principal autophagy triggers include starvation, pathogens, and stress. Autophagy plays also a pivotal role in immune response regulation, including immune cell differentiation, antigen presentation and the generation of T effector responses, the development of protective immunity against pathogens, and the coordination of immunometabolic signals. A plethora of studies propose that both impaired and overactive autophagic processes contribute to the pathogenesis of human disorders, including infections, cancer, atherosclerosis, autoimmune and neurodegenerative diseases. Autophagy has been also implicated in the development and progression of allergen-driven airway inflammation and remodeling. Here, we provide an overview of recent studies pertinent to the biology of autophagy and molecular pathways controlling its activation, we discuss autophagy-mediated beneficial and detrimental effects in animal models of allergic diseases and illuminate new advances on the role of autophagy in the pathogenesis of human asthma. We conclude contemplating the potential of targeting autophagy as a novel therapeutic approach for the management of allergic responses and linked asthmatic disease.

COVID-19: Lung-Centric Immunothrombosis

The respiratory tract is the major site of infection by SARS-CoV-2, the virus causing COVID-19. The pulmonary infection can lead to acute respiratory distress syndrome (ARDS) and ultimately, death. An excessive innate immune response plays a major role in the development of ARDS in COVID-19 patients. In this scenario, activation of lung epithelia and resident macrophages by the virus results in local cytokine production and recruitment of neutrophils. Activated neutrophils extrude a web of DNA-based cytoplasmic material containing antimicrobials referred to as neutrophil extracellular traps (NETs). While NETs are a defensive strategy against invading microbes, they can also serve as a nidus for accumulation of activated platelets and coagulation factors, forming thrombi. This immunothrombosis can result in occlusion of blood vessels leading to ischemic damage. Herein we address evidence in favor of a lung-centric immunothrombosis and suggest a lung-centric therapeutic approach to the ARDS of COVID-19.

Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.

Emerging role of the Hippo pathway in autophagy

Autophagy is a dynamic circulatory system that occurs in all eukaryotic cells. Cytoplasmic material is transported to lysosomes for degradation and recovery through autophagy. This provides energy and macromolecular precursors for cell renewal and homeostasis. The Hippo-YAP pathway has significant biological properties in controlling organ size, tissue homeostasis, and regeneration. Recently, the Hippo-YAP axis has been extensively referred to as the pathophysiological processes regulating autophagy. Understanding the cellular and molecular basis of these processes is crucial for identifying disease pathogenesis and novel therapeutic targets. Here we review recent findings from Drosophila models to organisms. We particularly emphasize the regulation between Hippo core components and autophagy, which is involved in normal cellular regulation and the pathogenesis of human diseases, and its application to disease treatment.

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30-40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans.

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30-40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans.

Blooming trees as a source of fine (< 5μm) aerosol particles

&lt;p&gt;During the blooming season of trees, pollen is an important component of the atmospheric aerosol, even in urban areas. Wind pollinated plants such as early flowering trees (e.g. birch, alder) release pollen grains in extremely large quantities. Once in the atmosphere pollen can impact human health and cloud formation (Sch&amp;#228;ppi et al. 1999, Pummer et al. 2012, Steiner et al. 2015). Intact pollen grains are rather large with geometrical diameters from 10-100 &amp;#956;m and therefore have short residence times in the atmosphere. However, it is known that under certain conditions (high humidity and after germination) pollen grains release cytoplasmic material including starch granules from their interior, commonly referred to as subpollen particles (SPP). Studies have shown that the cytoplasmic material contains cloud active substances and allergens (Steiner et al. 2015, Pummer et al. 2012, Basci et al. 2006). How and if this material becomes airborne and whether it distributes in the atmosphere is still an open question. Motivated by this question we took a detailed look at the particles shed from blooming catkins.&lt;/p&gt; &lt;p&gt;In this study freshly harvested branches with flowering catkins of different trees were put in an aerosol chamber. An Aerodynamic Particle Sizer (TSI Spectrometer 3321; 0.5 &amp;#8211; 20 &amp;#956;m) and a Cascade Impactor (Sioutas; 2.5 &amp;#956;m, 1.0 &amp;#956;m, 0.50 &amp;#956;m, 0.25 &amp;#956;m) were attached to the chamber to sample the released aerosol. The catkins were agitated with puffs of clean air to simulate wind. The aerodynamic diameters of the released particles were recorded and the filters of the impactor were analyzed with a Scanning Electron Microscope and a light microscope. We find that not only large pollen grains are released but also smaller particles. Up to 50% of all released particles were in the size range from (0.5 &amp;#8211; 5 &amp;#956;m). Additionally, we find that the aerodynamic diameter of pollen grains is in general smaller than their geometrical diameter. For instance, the aerodynamic diameter of pollen grains from birch is 30-70% smaller than the geometrical diameter.&lt;/p&gt; &lt;p&gt;&amp;#160;&lt;/p&gt; &lt;p&gt;References:&lt;br /&gt;Sch&amp;#228;ppi, G. F.; Taylor, P. E.; Pain, M. C.; Cameron, P. A.; Dent, A. W.; Staff, I. A. &amp; Suphioglu, C.; Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens.; Clinical and experimental allergy: journal of the British Society for Allergy and Clinical Immunology, 1999, 29, 633-641&lt;br /&gt;Pummer, B. G.; Bauer, H.; Bernardi, J.; Bleicher, S. &amp; Grothe, H.; Suspendable macromolecules are responsible for ice nucleation activity of birch and conifer pollen; Atmospheric Chemistry and Physics, Copernicus GmbH, 2012, 12, 2541-2550&lt;br /&gt;Steiner, A. L.; Brooks, S. D.; Deng, C.; Thornton, D. C. O.; Pendleton, M. W. &amp; Bryant, V.; Pollen as atmospheric cloud condensation nuclei; Geophysical research letters, Wiley Online Library, 2015, 42, 3596-3602&lt;br /&gt;Bacsi, A.; Choudhury, B. K.; Dharajiya, N.; Sur, S. &amp; Boldogh, I.; Subpollen particles: carriers of allergenic proteins and oxidases; Journal of Allergy and Clinical Immunology, Elsevier, 2006 , 118 , 844-850&lt;/p&gt;

CCN properties of pollen from selected wind pollinated plants

&lt;p&gt;Aerosol particles that act as cloud condensation nuclei (CCN) inuence cloud albedo and lifetime and thereby affect the planetary radiative balance. The indirect aerosol effect on climate is still one of the largest uncertainties and especially the role of biological particles is not yet well described. Pollen grains are primary biological particles that become airborne during the blooming season of plants. Pollen from wind pollinated plants represent a seasonally signifficant portion of the organic aerosol in the atmosphere. Intact pollen grains are rather large (10-100 &amp;#181;m) but under conditions of high humidity pollen grains have been shown to rupture and release cytoplasmic material including a large number of particles much smaller in size (0.5-5 &amp;#181;m).&lt;/p&gt;&lt;p&gt;In this study we extract soluble and insoluble material from several pollen samples (&lt;em&gt;Phleum,&lt;/em&gt; &lt;em&gt;Betula&lt;/em&gt;, &lt;em&gt;Artimesia&lt;/em&gt;, &lt;em&gt;Poa&lt;/em&gt;, &lt;em&gt;Corylus&lt;/em&gt; and &lt;em&gt;Ambrosia&lt;/em&gt;) and investigate the CCN activity of the extracts in a laboratory study. The main component of the experiment is the continuous-flow streamwise thermal-gradient cloud condensation nuclei counter (CCNC) from Droplet Measurement Technologies (DMT). The CCNC was calibrated with (NH&lt;sub&gt;4&lt;/sub&gt;)2SO&lt;sub&gt;4&lt;/sub&gt;. The activation behavior of (NH&lt;sub&gt;4&lt;/sub&gt;)2SO&lt;sub&gt;4&lt;/sub&gt; is theoretically well described by Kohler equation. For particles which consist of a multitude of organic components it is convenient to represent the chemical composition through the hygroscopicity parameter &amp;#954;. In the first part of the experiment, we determine the activation diameter at 5 different supersaturations and calculate the kappa parameter for all pollen samples. We find that the values fall in the range from 0.1-0.2. which is typical for particles composed of organic substances. Extracts from &lt;em&gt;Betula&lt;/em&gt; pollen show the highest hygroscopicity (&amp;#954; = 0.18), while extracts from &lt;em&gt;Artimesia&lt;/em&gt; exhibit the lowest hygroscopicity (&amp;#954; = 0.13). In the second part of the experiment we will also investigate the CCN activity of the insoluble material.&lt;/p&gt;

Alpha 6 integrins promote cytokinesis by regulating the expression of RSK2 and MKLP1

ABSTRACTThe integrin-mediated interaction of cells with components of the extracellular matrix (ECM) regulates many cellular processes including cell division. Cytokinesis is the last step of cell division and is critical for normal development and tissue homeostasis as it ensures the proper segregation of genetic and cytoplasmic material between daughter cells. Cytokinesis failure leads to defects in development and tissue differentiation, as well as tumorigenesis. Abscission of intercellular bridge that connects presumptive daughter cells is the last step of cell division. The mitotic kinesin-like protein 1 (MKLP1) plays a central role in positioning the abscission machinery. Here, we show that α6 integrins promote successful cytokinesis in salivary gland epithelial cells by regulating the expression of MKLP1. RNAi-mediated depletion of α6 integrins inhibits cytokinesis and the expression of MKLP1 and p90 ribosomal-S6-kinase 2 (RSK2). Depletion of RSK2 results in similar defects in cytokinesis and also inhibits the expression of MKLP1, suggesting that the expression of RSK2 is required downstream of integrins to promote MKLP1 expression and successful cytokinesis. RNAi-mediated depletion of RSK2 in embryonic salivary glands in organ culture also results in the inhibition of cytokinesis and MKLP1 expression, indicating the physiological significance of this pathway.