A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Freeze Substitution Accelerated via Agitation: New Prospects for Ultrastructural Studies of Lichen Symbionts and Their Extracellular Matrix.

(1) Background: Lichens, as an important part of the terrestrial ecosystem, attract the attention of various research disciplines. To elucidate their ultrastructure, transmission electron microscopy of resin-embedded samples is indispensable. Since most observations of lichen samples are generated via chemical fixation and processing at room temperature, they lack the rapid immobilization of live processes and are prone to preparation artefacts. To improve their preservation, cryoprocessing was tested in the past, but never widely implemented, not least because of an extremely lengthy protocol. (2) Methods: Here, we introduce an accelerated automated freeze substitution protocol with continuous agitation. Using the example of three lichen species, we demonstrate the preservation of the native state of algal photobionts and mycobionts in association with their extracellular matrix. (3) Results: We bring to attention the extent and the structural variability of the hyphae, the extracellular matrix and numerous crystallized metabolites. Our findings will encourage studies on transformation processes related to the compartmentation of lichen thalli. They include cryopreserved aspects of algal photobionts and observations of putative physiological relevance, such as the arrangement of numerous mitochondria within chloroplast pockets. (4) Conclusions: In summary, we present accelerated freeze substitution as a very useful tool for systematic studies of lichen ultrastructures.

Extracellular Vesicles and Particles Modulate Proton Secretion in a Model of Human Parietal Cells.

The secretion of extracellular vesicles and particles (EVPs) is an important mechanism of cellular communication. In this work, we demonstrate a functional role of EVPs in mechanisms regulating gastric acid secretion. HGT-1 cells were used as a model system to assess proton secretion. First, in order to prove EVP secretion by HGT-1 cells, EVPs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, Western blot, and cryo transmission electron microscopy. For examination of the potential role of EVPs in proton secretion, HGT-1 cells were treated with pharmacological EV-inhibitors, resulting in a reduction of histamine-induced proton secretion. To demonstrate the functional role of EVPs in the mechanism of proton secretion, EVP-conditioned supernatant was collected after stimulation of HGT-1 cells with histamine, fractionated, and subjected to an activity screening. The results revealed constituents of the HGT-1-derived secretome with an MW of >100 kDa (including EVPs) to modulate proton secretion, while smaller constituents had no effect. Finally, a dose-dependent modulatory effect on proton secretion of HGT-1 cells was demonstrated by isolated HGT-1-derived EVPs. Hence, this study presents first results on the potential function of EVPs as a previously undiscovered mechanism of regulation of gastric acid secretion by parietal cells.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

Hepatectomy-induced apoptotic extracellular vesicles stimulate neutrophils to secrete regenerative growth factors.

BACKGROUND & AIMS: Surgical resection of the cancerous tissue represents one of the few curative treatment options for neoplastic liver disease. Such partial hepatectomy (PHx) induces hepatocyte hyperplasia, which restores liver function. PHx is associated with bacterial translocation, leading to an immediate immune response involving neutrophils and macrophages, which are indispensable for the priming phase of liver regeneration. Additionally, PHx induces longer-lasting intrahepatic apoptosis. Herein, we investigated the effect of apoptotic extracellular vesicles (aEVs) on neutrophil function and their role in this later phase of liver regeneration. METHODS: A total of 124 patients undergoing PHx were included in this study. Blood levels of the apoptosis marker caspase-cleaved cytokeratin-18 (M30) and circulating aEVs were analyzed preoperatively and on the first and fifth postoperative days. Additionally, the in vitro effects of aEVs on the secretome, phenotype and functions of neutrophils were investigated. RESULTS: Circulating aEVs increased at the first postoperative day and were associated with higher concentrations of M30, which was only observed in patients with complete liver recovery. Efferocytosis of aEVs by neutrophils induced an activated phenotype (CD11bhighCD16highCD66bhighCD62Llow); however, classical inflammatory responses such as NETosis, respiratory burst, degranulation, or secretion of pro-inflammatory cytokines were not observed. Instead, efferocytosing neutrophils released various growth factors including fibroblast growth factor-2 and hepatocyte growth factor (HGF). Accordingly, we observed an increase of HGF-positive neutrophils after PHx and a correlation of plasma HGF with M30 levels. CONCLUSIONS: These data suggest that the clearance of PHx-induced aEVs leads to a population of non-inflammatory but regenerative neutrophils, which may support human liver regeneration. LAY SUMMARY: In this study, we show that the surgical removal of a diseased part of the liver triggers a specific type of programmed cell death in the residual liver tissue. This results in the release of vesicles from dying cells into the blood, where they are cleared by circulating immune cells. These respond by secreting hepatocyte growth factors that could potentially support the regeneration of the liver remnant.

Albumin-targeting of an oxaliplatin-releasing platinum(iv) prodrug results in pronounced anticancer activity due to endocytotic drug uptake in vivo.

Oxaliplatin is a very potent platinum(ii) drug which is frequently used in poly-chemotherapy schemes against advanced colorectal cancer. However, its benefit is limited by severe adverse effects as well as resistance development. Based on their higher tolerability, platinum(iv) prodrugs came into focus of interest. However, comparable to their platinum(ii) counterparts they lack tumor specificity and are frequently prematurely activated in the blood circulation. With the aim to exploit the enhanced albumin consumption and accumulation in the malignant tissue, we have recently developed a new albumin-targeted prodrug, which supposed to release oxaliplatin in a highly tumor-specific manner. In more detail, we designed a platinum(iv) complex containing two maleimide moieties in the axial position (KP2156), which allows selective binding to the cysteine 34. In the present study, diverse cell biological and analytical tools such as laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), isotope labeling, and nano-scale secondary ion mass spectrometry (NanoSIMS) were employed to better understand the in vivo distribution and activation process of KP2156 (in comparison to free oxaliplatin and a non-albumin-binding succinimide analogue). KP2156 forms very stable albumin adducts in the bloodstream resulting in a superior pharmacological profile, such as distinctly prolonged terminal excretion half-life and enhanced effective platinum dose (measured by ICP-MS). The albumin-bound drug is accumulating in the malignant tissue, where it enters the cancer cells via clathrin- and caveolin-dependent endocytosis, and is activated by reduction to release oxaliplatin. This results in profound, long-lasting anticancer activity of KP2156 against CT26 colon cancer tumors in vivo based on cell cycle arrest and apoptotic cell death. Summarizing, albumin-binding of platinum(iv) complexes potently enhances the efficacy of oxaliplatin therapy and should be further developed towards clinical phase I trials.

Recently photoassimilated carbon and fungus-delivered nitrogen are spatially correlated in the ectomycorrhizal tissue of Fagus sylvatica.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15 N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13 CO2 atmosphere. We analysed the short-term distribution of 13 C and 15 N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13 C to root parts that received more 15 N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13 C and 15 N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Anaerobic Sulfur Oxidation Underlies Adaptation of a Chemosynthetic Symbiont to Oxic-Anoxic Interfaces.

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology. Here, by applying omics, Raman microspectroscopy, and stable isotope labeling, we investigated the effect of oxygen on "Candidatus Thiosymbion oneisti." Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated under oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation, and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand oxidative stress, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that "Ca. T. oneisti" may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation. IMPORTANCE Chemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One longstanding hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of "Candidatus Thiosymbion oneisti," a gammaproteobacterium which lives attached to marine nematodes inhabiting shallow-water sand. Surprisingly, sulfur oxidation genes were upregulated under anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

Nano-scale imaging of dual stable isotope labeled oxaliplatin in human colon cancer cells reveals the nucleolus as a putative node for therapeutic effect.

Oxaliplatin shows a superior clinical activity in colorectal cancer compared to cisplatin. Nevertheless, the knowledge about its cellular distribution and the mechanisms responsible for the different range of oxaliplatin-responsive tumors is far from complete. In this study, we combined highly sensitive element specific and isotope selective imaging by nanometer-scale secondary ion mass spectrometry (NanoSIMS) with transmission electron microscopy to investigate the subcellular accumulation of oxaliplatin in three human colon cancer cell lines (SW480, HCT116 wt, HCT116 OxR). Oxaliplatin bearing dual stable isotope labeled moieties, i.e. 2H-labeled diaminocyclohexane (DACH) and 13C-labeled oxalate, were applied for comparative analysis of the subcellular distribution patterns of the central metal and the ligands. In all the investigated cell lines, oxaliplatin was found to have a pronounced tendency for cytoplasmic aggregation in single membrane bound organelles, presumably related to various stages of the endocytic pathway. Moreover, nuclear structures, heterochromatin and in particular nucleoli, were affected by platinum-drug exposure. In order to explore the consequences of oxaliplatin resistance, subcellular drug distribution patterns were investigated in a pair of isogenic malignant cell lines with distinct levels of drug sensitivity (HCT116 wt and HCT116 OxR, the latter with acquired resistance to oxaliplatin). The subcellular platinum distribution was found to be similar in both cell lines, with only slightly higher accumulation in the sensitive HCT116 wt cells which is inconsistent with the resistance factor of more than 20-fold. Instead, the isotopic analysis revealed a disproportionally high accumulation of the oxalate ligand in the resistant cell line.

Interaction with Ribosomal Proteins Accompanies Stress Induction of the Anticancer Metallodrug BOLD-100/KP1339 in the Endoplasmic Reticulum.

The ruthenium-based anticancer agent BOLD-100/KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target-profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells.

Deficiency of Cathelicidin-related Antimicrobial Peptide Promotes Skin Papillomatosis in Mus musculus Papillomavirus 1-infected Mice.

Cathelicidins have been reported to inhibit human papillomavirus infection in vitro; however, nothing is known about their activity in vivo. In this study, experimental skin infection with Mus musculus papillomavirus 1 resulted in robust development of cutaneous papillomas in cyclosporine A-treated C57BL/6J mice deficient for the murine cathelicidin-related antimicrobial peptide (CRAMP), in contrast to wild-type controls. Analysis of the underlying mechanisms revealed moderate disruption of virion integrity and lack of interference with viral entry and intracellular trafficking by a synthetic CRAMP peptide. Differences in the immune response to Mus musculus papillomavirus 1 infection were observed between CRAMP-deficient and wild-type mice. These included a stronger reduction in CD4+ and CD8+ T-cell numbers in infected skin, and lack of Mus musculus papillomavirus 1-specific neutralizing antibodies in response to cyclosporine A in the absence of endogenous CRAMP. CRAMP has modest direct anti-papillomaviral effects in vitro, but exerts protective functions against Mus musculus papillomavirus 1 skin infection and disease development in vivo, primarily by modulation of cellular and humoral immunity.

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s.

Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.

Agitation Modules: Flexible Means to Accelerate Automated Freeze Substitution.

For ultrafast fixation of biological samples to avoid artifacts, high-pressure freezing (HPF) followed by freeze substitution (FS) is preferred over chemical fixation at room temperature. After HPF, samples are maintained at low temperature during dehydration and fixation, while avoiding damaging recrystallization. This is a notoriously slow process. McDonald and Webb demonstrated, in 2011, that sample agitation during FS dramatically reduces the necessary time. Then, in 2015, we (H.G. and S.R.) introduced an agitation module into the cryochamber of an automated FS unit and demonstrated that the preparation of algae could be shortened from days to a couple of hours. We argued that variability in the processing, reproducibility, and safety issues are better addressed using automated FS units. For dissemination, we started low-cost manufacturing of agitation modules for two of the most widely used FS units, the Automatic Freeze Substitution Systems, AFS(1) and AFS2, from Leica Microsystems, using three dimensional (3D)-printing of the major components. To test them, several labs independently used the modules on a wide variety of specimens that had previously been processed by manual agitation, or without agitation. We demonstrate that automated processing with sample agitation saves time, increases flexibility with respect to sample requirements and protocols, and produces data of at least as good quality as other approaches.

Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.

Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host.

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

Novel mesostructured inclusions in the epidermal lining of Artemia franciscana ovisacs show optical activity.

BACKGROUND: Biomineralization, e.g., in sea urchins or mollusks, includes the assembly of mesoscopic superstructures from inorganic crystalline components and biopolymers. The resulting mesocrystals inspire biophysicists and material scientists alike, because of their extraordinary physical properties. Current efforts to replicate mesocrystal synthesis in vitro require understanding the principles of their self-assembly in vivo. One question, not addressed so far, is whether intracellular crystals of proteins can assemble with biopolymers into functional mesocrystal-like structures. During our electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we found initial evidence of such proteinaceous mesostructures. RESULTS: EM preparations with high-pressure freezing and accelerated freeze substitution revealed an extraordinary intracellular source of mesostructured inclusions in both the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana. Confocal reflection microscopy not only confirmed our finding; it also revealed reflective, light dispersing activity of these flake-like structures, their positioning and orientation with respect to the ovisac inside. Both the striation of alternating electron dense and electron-lucent components and the sharp edges of the flakes indicate self-assembly of material of yet unknown origin under supposed participation of crystallization. However, selected area electron diffraction could not verify the status of crystallization. Energy dispersive X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion, and the almost complete absence of elements that are typically involved in inorganic crystallization. This rise in nitrogen could possibility be related to higher package density of proteins, achieved by mesostructure assembly. CONCLUSIONS: The ovisac lining of A. franciscana is endowed with numerous mesostructured inclusions that have not been previously reported. We hypothesize that their self-assembly was from proteinaceous polycrystalline units and carbohydrates. These mesostructured flakes displayed active optical properties, as an umbrella-like, reflective cover of the ovisac, which suggests a functional role in the reproduction of A. franciscana. In turn, studies into ovisac mesostructured inclusions could help to optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an in vivo model system for studying mesostructure formation.

Novel p53-dependent anticancer strategy by targeting iron signaling and BNIP3L-induced mitophagy.

This study identifies BNIP3L as the key regulator of p53-dependent cell death mechanism in colon cancer cells targeted by the novel gallium based anticancer drug, KP46. KP46 specifically accumulated into mitochondria where it caused p53-dependent morphological and functional damage impairing mitochondrial dynamics and bioenergetics. Furthermore, competing with iron for cellular uptake, KP46 lowered the intracellular labile iron pools and intracellular heme. Accordingly, p53 accumulated in the nucleus where it activated its transcriptional target BNIP3L, a BH3 only domain protein with functions in apoptosis and mitophagy. Upregulated BNIP3L sensitized the mitochondrial permeability transition and strongly induced PARKIN-mediated mitochondrial clearance and cellular vacuolization. Downregulation of BNIP3L entirely rescued cell viability caused by exposure of KP46 for 24 hours, confirming that early induced cell death was regulated by BNIP3L. Altogether, targeting BNIP3L in wild-type p53 colon cancer cells is a novel anticancer strategy activating iron depletion signaling and the mitophagy-related cell death pathway.