GPRC5C regulates the composition of cilia in the olfactory system.

BACKGROUND: Olfactory sensory neurons detect odourants via multiple long cilia that protrude from their dendritic endings. The G protein-coupled receptor GPRC5C was identified as part of the olfactory ciliary membrane proteome, but its function and localization is unknown. RESULTS: High-resolution confocal and electron microscopy revealed that GPRC5C is located at the base of sensory cilia in olfactory neurons, but not in primary cilia of immature neurons or stem cells. Additionally, GPRC5C localization in sensory cilia parallels cilia formation and follows the formation of the basal body. In closer examination, GPRC5C was found in the ciliary transition zone. GPRC5C deficiency altered the structure of sensory cilia and increased ciliary layer thickness. However, primary cilia were unaffected. Olfactory sensory neurons from Gprc5c-deficient mice exhibited altered localization of olfactory signalling cascade proteins, and of ciliary phosphatidylinositol-4,5-bisphosphat. Sensory neurons also exhibited increased neuronal activity as well as altered mitochondrial morphology, and knockout mice had an improved ability to detect food pellets based on smell. CONCLUSIONS: Our study shows that GPRC5C regulates olfactory cilia composition and length, thereby controlling odour perception.

Laboratory-Based Correlative Soft X-ray and Fluorescence Microscopy in an Integrated Setup.

Correlative microscopy is a powerful technique that combines the advantages of multiple imaging modalities to achieve a comprehensive understanding of investigated samples. For example, fluorescence microscopy provides unique functional contrast by imaging only specifically labeled components, especially in biological samples. However, the achievable structural information on the sample in its full complexity is limited. Here, the intrinsic label-free carbon contrast of water window soft X-ray microscopy can complement fluorescence images in a correlative approach ultimately combining nanoscale structural resolution with functional contrast. However, soft X-ray microscopes are complex and elaborate, and are usually installed on large-scale synchrotron radiation sources due to the demanding photon flux requirements. Yet, with modern high-power lasers it has become possible to generate sufficient photon flux from laser-produced plasmas, thus enabling laboratory-based setups. Here, we present a compact table-top soft X-ray microscope with an integrated epifluorescence modality for "in situ" correlative imaging. Samples remain in place when switching between modalities, ensuring identical measurement conditions and avoiding sample alteration or destruction. We demonstrate our new method by multimodal images of several exemplary samples ranging from nanoparticles to various multicolor labeled cell types. A structural resolution of down to 50 nm was reached.

Different lanthanide elements induce strong gene expression changes in a lanthanide-accumulating methylotroph.

IMPORTANCE: Since its discovery, Ln-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding Ln recycling and circular economy. The physiological role of Ln is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) Ln affect metabolism have rarely been done. We noticed unexpectedly pronounced changes in gene expression caused by different Ln supplementation. Our research suggests that strain RH AL1 distinguishes different Ln elements and that the effect of Ln reaches into many aspects of metabolism, for instance, chemotaxis, motility, and polyhydroxyalkanoate metabolism. Our findings regarding Ln accumulation suggest a distinction between individual Ln elements and provide insights relating to intracellular Ln homeostasis. Understanding comprehensively how microbes distinguish and handle different Ln elements is key for turning knowledge into application regarding Ln-centered biometallurgy.

Comparative genomics hints at dispensability of multiple essential genes in two Escherichia coli L-form strains.

Despite the critical role of bacterial cell walls in maintaining cell shapes, certain environmental stressors can induce the transition of many bacterial species into a wall-deficient state called L-form. Long-term induced Escherichia coli L-forms lose their rod shape and usually hold significant mutations that affect cell division and growth. Besides this, the genetic background of L-form bacteria is still poorly understood. In the present study, the genomes of two stable L-form strains of E. coli (NC-7 and LWF+) were sequenced and their gene mutation status was determined and compared with their parental strains. Comparative genomic analysis between two L-forms reveals both unique adaptions and common mutated genes, many of which belong to essential gene categories not involved in cell wall biosynthesis, indicating that L-form genetic adaptation impacts crucial metabolic pathways. Missense variants from L-forms and Lenski's long-term evolution experiment (LTEE) were analyzed in parallel using an optimized DeepSequence pipeline to investigate predicted mutation effects (α) on protein functions. We report that the two L-form strains analyzed display a frequency of 6-10% (0% for LTEE) in mutated essential genes where the missense variants have substantial impact on protein functions (α<0.5). This indicates the emergence of different survival strategies in L-forms through changes in essential genes during adaptions to cell wall deficiency. Collectively, our results shed light on the detailed genetic background of two E. coli L-forms and pave the way for further investigations of the gene functions in L-form bacterial models.

The choline-binding proteins PspA, PspC, and LytA of Streptococcus pneumoniae and their interaction with human endothelial and red blood cells.

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia and meningitis. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries. Here, we focused on three choline-binding proteins (CBPs), i.e., PspC, PspA, and LytA. These pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy, and functional quantitative assays, targeting biofilm formation and the hemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC, or pspA genes to adhere to surfaces. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the hemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of each CPB for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

The role of pneumococcal extracellular vesicles on the pathophysiology of the kidney disease hemolytic uremic syndrome.

Streptococcus pneumoniae-induced hemolytic uremic syndrome (Sp-HUS) is a kidney disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. This disease is frequently underdiagnosed and its pathophysiology is poorly understood. In this work, we compared clinical strains, isolated from infant Sp-HUS patients, with a reference pathogenic strain D39, for host cytotoxicity and further explored the role of Sp-derived extracellular vesicles (EVs) in the pathogenesis of an HUS infection. In comparison with the wild-type strain, pneumococcal HUS strains caused significant lysis of human erythrocytes and increased the release of hydrogen peroxide. Isolated Sp-HUS EVs were characterized by performing dynamic light-scattering microscopy and proteomic analysis. Sp-HUS strain released EVs at a constant concentration during growth, yet the size of the EVs varied and several subpopulations emerged at later time points. The cargo of the Sp-HUS EVs included several virulence factors at high abundance, i.e., the ribosomal subunit assembly factor BipA, the pneumococcal surface protein A, the lytic enzyme LytC, several sugar utilization, and fatty acid synthesis proteins. Sp-HUS EVs strongly downregulated the expression of the endothelial surface marker platelet endothelial cell adhesion molecule-1 and were internalized by human endothelial cells. Sp-HUS EVs elicited the release of pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6) and chemokines (CCL2, CCL3, CXCL1) by human monocytes. These findings shed new light on the overall function of Sp-EVs, in the scope of infection-mediated HUS, and suggest new avenues of research for exploring the usefulness of Sp-EVs as therapeutic and diagnostic targets. IMPORTANCE Streptococcus pneumoniae-associated hemolytic uremic syndrome (Sp-HUS) is a serious and underdiagnosed deadly complication of invasive pneumococcal disease. Despite the introduction of the pneumococcal vaccine, cases of Sp-HUS continue to emerge, especially in children under the age of 2. While a lot has been studied regarding pneumococcal proteins and their role on Sp-HUS pathophysiology, little is known about the role of extracellular vesicles (EVs). In our work, we isolate and initially characterize EVs from a reference pathogenic strain (D39) and a strain isolated from a 2-year-old patient suffering from Sp-HUS. We demonstrate that despite lacking cytotoxicity toward human cells, Sp-HUS EVs are highly internalized by endothelial cells and can trigger cytokine and chemokine production in monocytes. In addition, this work specifically highlights the distinct morphological characteristics of Sp-HUS EVs and their unique cargo. Overall, this work sheds new light into potentially relevant players contained in EVs that might elucidate about pneumococcal EVs biogenesis or pose as interesting candidates for vaccine design.

Long-term depression in neurons involves temporal and ultra-structural dynamics of phosphatidylinositol-4,5-bisphosphate relying on PIP5K, PTEN and PLC.

Synaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP2 signals during induction of long-term depression (LTD). During the first minutes PIP2 rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP2 accumulation. The transiently increased PIP2 signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP2 degradation provides timely termination of PIP2 cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP2 during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP2 dynamics.

Light and prey influence the abundances of two rhodopsins in the dinoflagellate Oxyrrhis marina.

Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina. The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii. The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii. Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled.

Local Magnetic Hyperthermia and Systemic Gemcitabine/Paclitaxel Chemotherapy Triggers Neo-Angiogenesis in Orthotopic Pancreatic Tumors without Involvement of Auto/Paracrine Tumor Cell VEGF Signaling and Hypoxia.

There is a growing interest in exploring the therapeutically mediated modulation of tumor vascularization of pancreatic cancer, which is known for its poorly perfused tumor microenvironment limiting the delivery of therapeutic agents to the tumor site. Here, we assessed how magnetic hyperthermia in combination with chemotherapy selectively affects growth, the vascular compartment of tumors, and the presence of tumor cells expressing key regulators of angiogenesis. To that purpose, a orthotopic PANC-1 (fluorescent human pancreatic adenocarcinoma) mouse tumor model (Rj:Athym-Foxn1nu/nu) was used. Magnetic hyperthermia was applied alone or in combination with systemic chemotherapy (gemcitabine 50 mg per kg body weight, nab-pacitaxel 30 mg/kg body weight) on days 1 and 7 following magnetic nanoparticle application (dose: 1 mg per 100 mm3 of tumor). We used ultrasound imaging, immunohistochemistry, multi-spectral optoacoustic tomography (MSOT), and hematology to assess the biological parameters mentioned above. We found that magnetic hyperthermia in combination with gemcitabine/paclitaxel chemotherapy was able to impact tumor growth (decreased volumes and Ki67 expression) and to trigger neo-angiogenesis (increased small vessel diameter) as a result of the therapeutically mediated cell damages/stress in tumors. The applied stressors activated specific pro-angiogenic mechanisms, which differed from those seen in hypoxic conditions involving HIF-1α, since (a) treated tumors showed a significant decrease of cells expressing VEGF, CD31, HIF-1α, and neuropilin-1; and (b) the relative tumor blood volume and oxygen level remained unchanged. Neo-angiogenesis seems to be the result of the activation of cell stress pathways, like MAPK pathways (high number of pERK-expressing tumor cells). In the long term, the combination of magnetic hyperthermia and chemotherapy could potentially be applied to transiently modulate tumor angiogenesis and to improve drug accessibility during oncologic therapies of pancreatic cancer.

Impact of in vitro digested zinc oxide nanoparticles on intestinal model systems.

BACKGROUND: Zinc oxide nanoparticles (ZnO NP) offer beneficial properties for many applications, especially in the food sector. Consequently, as part of the human food chain, they are taken up orally. The toxicological evaluation of orally ingested ZnO NP is still controversial. In addition, their physicochemical properties can change during digestion, which leads to an altered biological behaviour. Therefore, the aim of our study was to investigate the fate of two different sized ZnO NP (< 50 nm and < 100 nm) during in vitro digestion and their effects on model systems of the intestinal barrier. Differentiated Caco-2 cells were used in mono- and coculture with mucus-producing HT29-MTX cells. The cellular uptake, the impact on the monolayer barrier integrity and cytotoxic effects were investigated after 24 h exposure to 123-614 µM ZnO NP. RESULTS: In vitro digested ZnO NP went through a morphological and chemical transformation with about 70% free zinc ions after the intestinal phase. The cellular zinc content increased dose-dependently up to threefold in the monoculture and fourfold in the coculture after treatment with digested ZnO NP. This led to reactive oxygen species but showed no impact on cellular organelles, the metabolic activity, and the mitochondrial membrane potential. Only very small amounts of zinc (< 0.7%) reached the basolateral area, which is due to the unmodified transepithelial electrical resistance, permeability, and cytoskeletal morphology. CONCLUSIONS: Our results reveal that digested and, therefore, modified ZnO NP interact with cells of an intact intestinal barrier. But this is not associated with serious cell damage.

Artificial Digestion of Polydisperse Copper Oxide Nanoparticles: Investigation of Effects on the Human In Vitro Intestinal Co-Culture Model Caco-2/HT29-MTX.

Copper oxide nanoparticles (CuO-NP) are increasingly used in consumer-related products, which may result in increased oral ingestion. Digestion of particles can change their physicochemical properties and toxicity. Therefore, our aim was to simulate the gastrointestinal tract using a static in vitro digestion model. Toxic properties of digested and undigested CuO-NP were compared using an epithelial mono-culture (Caco-2) and a mucus-secreting co-culture model (Caco-2/HT29-MTX). Effects on intestinal barrier integrity, permeability, cell viability and apoptosis were analyzed. CuO-NP concentrations of 1, 10 and 100 µg mL-1 were used. Particle characterization by dynamic light scattering and transmission electron microscopy showed similar mean particle sizes before and after digestion, resulting in comparable delivered particle doses in vitro. Only slight effects on barrier integrity and cell viability were detected for 100 µg mL-1 CuO-NP, while the ion control CuCl2 always caused significantly higher adverse effects. The utilized cell models were not significantly different. In summary, undigested and digested CuO-NP show comparable effects on the mono-/co-cultures, which are weaker than those of copper ions. Only in the highest concentration, CuO-NP showed weak effects on barrier integrity and cell viability. Nevertheless, a slightly increased apoptosis rate indicates existing cellular stress, which gives reason for further investigations.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina.

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2-7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0-7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Tumor-derived exosomes: Key players in non-small cell lung cancer metastasis and their implication for targeted therapy.

Exosomes represent extracellular vesicles of endocytic origin ranging from 30 to 100 nm that are released by most of eukaryotic cells and can be found in body fluids. These vesicles in carrying DNA, RNA, microRNA (miRNA), Long noncoding RNA, proteins, and lipids serve as intercellular communicators. Due to their role in crosstalk between tumor cells and mesenchymal stroma cells, they are vital for tumor growth, progression, and anticancer drug resistance. Lung cancer is a global leading cause of cancer-related deaths with 5-year survival rates of about 7% in patients with distant metastasis. Although the implementation of targeted therapy has improved the clinical outcome of nonsmall cell lung cancer, drug resistance remains a major obstacle. Lung tumor-derived exosomes (TDEs) conveying molecular information from tumor cells to their neighbor cells or cells at distant sites of the body activate the tumor microenvironment (TME) and facilitate tumor metastasis. Exosomal miRNAs are also considered as noninvasive biomarkers for early diagnosis of lung cancer. This review summarizes the influence of lung TDEs on the TME and metastasis. Their involvement in targeted therapy resistance and potential clinical applications are discussed. Additionally, challenges encountered in the development of exosome-based therapeutic strategies are addressed.

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis.

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis.

The fungivorous amoeba Protostelium aurantium targets redox homeostasis and cell wall integrity during intracellular killing of Candida parapsilosis.

Predatory interactions among microbes are major evolutionary driving forces for biodiversity. The fungivorous amoeba Protostelium aurantium has a wide fungal food spectrum including foremost pathogenic members of the genus Candida. Here we show that upon phagocytic ingestion by the amoeba, Candida parapsilosis is confronted with an oxidative burst and undergoes lysis within minutes of processing in acidified phagolysosomes. On the fungal side, a functional genomic approach identified copper and redox homeostasis as primary targets of amoeba predation, with the highly expressed copper exporter gene CRP1 and the peroxiredoxin gene PRX1 contributing to survival when encountered with P. aurantium. The fungicidal activity was largely retained in intracellular vesicles of the amoebae. Following their isolation, the content of these vesicles induced immediate killing and lysis of C. parapsilosis in vitro. Proteomic analysis identified 56 vesicular proteins from P. aurantium. Although completely unknown proteins were dominant, many of them could be categorised as hydrolytic enzymes targeting the fungal cell wall, indicating that fungal cell wall structures are under selection pressure by predatory phagocytes in natural environments. TAKE AWAY: The amoeba Protostelium aurantium feeds on fungi, such as Candida parapsilosis. Ingested yeast cells are exposed to reactive oxygen species. A copper exporter and a peroxiredoxin contribute to fungal defence. Yeast cells undergo intracellular lysis. Lysis occurs via a cocktail of hydrolytic enzymes from intracellular vesicles.

DASH cryptochrome 1, a UV-A receptor, balances the photosynthetic machinery of Chlamydomonas reinhardtii.

Drosophila, Arabidopsis, Synechocystis, Homo (DASH) cryptochromes belong to the cryptochrome/photolyase family and can act as DNA repair enzymes. In bacteria and fungi, they also can play regulatory roles, but in plants their biological functions remain elusive. Here, we characterize CRY-DASH1 from the green alga Chlamydomonas reinhardtii. We perform biochemical and in vitro photochemical analysis. For functional characterization, a knock-out mutant of cry-dash1 is used. CRY-DASH1 protein is localized in the chloroplast and accumulates at midday. Although the photoautotrophic growth of the mutant is significantly reduced compared to the wild-type (WT), the mutant has increased levels of photosynthetic pigments and a higher maximum photochemical efficiency of photosystem II (PS II). Hyper-stacking of thylakoid membranes occurs together with an increase in proteins of the PS II reaction center, D1 and its antenna CP43, but not of their transcripts. CRY-DASH1 binds fully reduced flavin adenine dinucleotide and the antenna 5,10-methenyltetrahydrofolate, leading to an absorption peak in the UV-A range. Supplementation of white light with UV-A increases photoautotrophic growth of the WT but not of the cry-dash1 mutant. These results suggest a balancing function of CRY-DASH1 in the photosynthetic machinery and point to its role as a photoreceptor for the UV-A range separated from the absorption of photosynthetic pigments.

Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells.

Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

Two Distinct Bacterial Biofilm Components Trigger Metamorphosis in the Colonial Hydrozoan Hydractinia echinata.

In marine environments, the bacterially induced metamorphosis of larvae is a widespread cross-kingdom communication phenomenon that is critical for the persistence of many marine invertebrates. However, the majority of inducing bacterial signals and underlying cellular mechanisms remain enigmatic. The marine hydroid Hydractinia echinata is a well-known model system for investigating bacterially stimulated larval metamorphosis, as larvae transform into the colonial adult stage within 24 h of signal detection. Although H. echinata has served as a cell biological model system for decades, the identity and influence of bacterial signals on the morphogenic transition remained largely unexplored. Using a bioassay-guided analysis, we first determined that specific bacterial (lyso)phospholipids, naturally present in bacterial membranes and vesicles, elicit metamorphosis in Hydractinia larvae in a dose-response manner. Lysophospholipids, as single compounds or in combination (50 µM), induced metamorphosis in up to 50% of all larvae within 48 h. Using fluorescence-labeled bacterial phospholipids, we demonstrated that phospholipids are incorporated into the larval membranes, where interactions with internal signaling cascades are proposed to occur. Second, we identified two structurally distinct exopolysaccharides of bacterial biofilms, the new Rha-Man polysaccharide from Pseudoalteromonas sp. strain P1-9 and curdlan from Alcaligenes faecalis, to induce metamorphosis in up to 75% of tested larvae. We also found that combinations of (lyso)phospholipids and curdlan induced transformation within 24 h, thereby exceeding the morphogenic activity observed for single compounds and bacterial biofilms. Our results demonstrate that two structurally distinct, bacterium-derived metabolites converge to induce high transformation rates of Hydractinia larvae and thus may help ensure optimal habitat selection. IMPORTANCE Bacterial biofilms profoundly influence the recruitment and settlement of marine invertebrates, critical steps for diverse marine processes such as the formation of coral reefs, the maintenance of marine fisheries, and the fouling of submerged surfaces. However, the complex composition of biofilms often makes the characterization of individual signals and regulatory mechanisms challenging. Developing tractable model systems to characterize these coevolved interactions is the key to understanding fundamental processes in evolutionary biology. Here, we characterized two types of bacterial signaling molecules, phospholipids and polysaccharides, that induce the morphogenic transition. We then analyzed their abundance and combinatorial activity. This study highlights the general importance of multiple bacterial signal converging activity in development-related cross-kingdom signaling and poses the question of whether complex lipids and polysaccharides are general metamorphic cues for cnidarian larvae.

Intracellular persistence of Staphylococcus aureus in endothelial cells is promoted by the absence of phenol-soluble modulins.

A large proportion of clinical S. aureus isolates that carry an inactive Agr system are associated with persistent infection that is difficult to treat. Once S. aureus is inside the bloodstream, it can cross the endothelial barrier and invade almost every organ in the human body. Endothelial cells can either be lysed by this pathogen or they serve as a niche for its intracellular long-term survival. Following phagocytosis, several vesicles such as phagosomes and autophagosomes, target intracellular S. aureus for elimination. S. aureus can escape from these vesicles into the host cytoplasm through the activation of phenol-soluble modulins (PSMs) αß. Thereafter, it replicates and lyses the host cell to disseminate to adjacent tissues. Herein we demonstrate that staphylococcal strains which lack the expression of PSMs employ an alternative pathway to better persist within endothelial cells. The intracellular survival of S. aureus is associated with the co-localization of the autophagy marker LC3. In cell culture infection models, we found that the absence of psmαß decreased the host cell lysis and increased staphylococcal long-term survival. This study explains the positive selection of agr-negative strains that lack the expression of psmαß in chronic infection due to their advantage in surviving and evading the clearance system of the host.