Age-specific and compartment-dependent changes in mitochondrial homeostasis and cytoplasmic viscosity in mouse peripheral neurons.

Mitochondria are dynamic bioenergetic hubs that become compromised with age. In neurons, declining mitochondrial axonal transport has been associated with reduced cellular health. However, it is still unclear to what extent the decline of mitochondrial transport and function observed during ageing are coupled, and if somal and axonal mitochondria display compartment-specific features that make them more susceptible to the ageing process. It is also not known whether the biophysical state of the cytoplasm, thought to affect many cellular functions, changes with age to impact mitochondrial trafficking and homeostasis. Focusing on the mouse peripheral nervous system, we show that age-dependent decline in mitochondrial trafficking is accompanied by reduction of mitochondrial membrane potential and intramitochondrial viscosity, but not calcium buffering, in both somal and axonal mitochondria. Intriguingly, we observe a specific increase in cytoplasmic viscosity in the neuronal cell body, where mitochondria are most polarised, which correlates with decreased cytoplasmic diffusiveness. Increasing cytoplasmic crowding in the somatic compartment of DRG neurons grown in microfluidic chambers reduces mitochondrial axonal trafficking, suggesting a mechanistic link between the regulation of cytoplasmic viscosity and mitochondrial dynamics. Our work provides a reference for studying the relationship between neuronal mitochondrial homeostasis and the viscoelasticity of the cytoplasm in a compartment-dependent manner during ageing.

Pten knockout in mouse preosteoblasts leads to changes in bone turnover and strength.

Bone development and remodeling are controlled by the phosphoinositide-3-kinase (Pi3k) signaling pathway. We investigated the effects of downregulation of phosphatase and tensin homolog (Pten), a negative regulator of Pi3k signaling, in a mouse model of Pten deficiency in preosteoblasts. We aimed to identify mechanisms that are involved in the regulation of bone turnover and are linked to bone disorders. Femora, tibiae, and bone marrow stromal cells (BMSCs) isolated from mice with a conditional deletion of Pten (Pten cKO) in Osterix/Sp7-expressing osteoprogenitor cells were compared to Cre-negative controls. Bone phenotyping was performed by µCT measurements, bone histomorphometry, quantification of bone turnover markers CTX and procollagen type 1 N propeptide (P1NP), and three-point bending test. Proliferation of BMSCs was measured by counting nuclei and Ki-67-stained cells. In vitro, osteogenic differentiation capacity was determined by ALP staining, as well as by detecting gene expression of osteogenic markers. BMSCs from Pten cKO mice were functionally different from control BMSCs. Osteogenic markers were increased in BMSCs derived from Pten cKO mice, while Pten protein expression was lower and Akt phosphorylation was increased. We detected a higher trabecular bone volume and an altered cortical bone morphology in Pten cKO bones with a progressive decrease in bone and tissue mineral density. Pten cKO bones displayed fewer osteoclasts and more osteoblasts (P = .00095) per trabecular bone surface and a higher trabecular bone formation rate. Biomechanical analysis revealed a significantly higher bone strength (P = .00012 for males) and elasticity of Pten cKO femora. On the cellular level, both proliferation and osteogenic differentiation capacity of Pten cKO BMSCs were significantly increased compared to controls. Our findings suggest that Pten knockout in osteoprogenitor cells increases bone stability and elasticity by increasing trabecular bone mass and leads to increased proliferation and osteogenic differentiation of BMSCs.

[Implementation of treatment by enzymatic debridement in burns: results of an interprofessional German-speaking expert workshop]. / Implementierung der Behandlung durch enzymatisches Debridement bei Verbrennungen ­ Ergebnisse eines interprofessionellen, deutschsprachigen Expertenworkshops.

INTRODUCTION: Since its introduction in 2013 Bromelain-based Enzymatic Debridement (ED) is increasingly used in burn centers. Published evidence shows its efficiency in eschar removal as well as a superiority in blood loss and necessity of further surgical procedures compared to standard-of-care. While the procedure is safe and shows reliable results in experienced hands, some practical and logistical issues must be challenged that are not described sufficiently in available literature. METHOD: A multi-professional panel, consisting of experienced users of ED from German-speaking burn units has been invited to an expert workshop. Topics concerning indication, definition of treatment pathways, practical issues, post-treatment and handling of complications have been coordinated in advance to allow discussion during the workshop. RESULTS: To each topic practical recommendations were developed and consented. Summarizing key messages have been additionally highlighted. They aim on helping to achieve optimal results after establishing the technique by new users as well as optimizing results by experienced users. Amongst others, the resulting recommendations deal with indications for ED beyond the classic domain, different treatment pathways depending on burn depth and primary result after ED with adapted post-treatment, management of treatment failure and implementation of infrastructural conditions. DISCUSSION: While efficiency of ED as well as superiority in some aspects of treatment of burn wounds could be shown in available literature, user-oriented recommendations for practical implementation are scarce. Although the recommendations and experts opinions published here are only partly evidenced based, they are still based on the pooled experienced of the panelists that easily outnumbers the cases published in literature so far and allow valuable support for a successful implementation of the technique.

Infection Patterns and Fitness Effects of Rickettsia and Sodalis Symbionts in the Green Lacewing Chrysoperla carnea.

Endosymbionts are widely distributed in insects and can strongly affect their host ecology. The common green lacewing (Chrysoperla carnea) is a neuropteran insect which is widely used in biological pest control. However, their endosymbionts and their interactions with their hosts have not been very well studied. Therefore, we screened for endosymbionts in natural and laboratory populations of Ch. carnea using diagnostic PCR amplicons. We found the endosymbiont Rickettsia to be very common in all screened natural and laboratory populations, while a hitherto uncharacterized Sodalis strain was found only in laboratory populations. By establishing lacewing lines with no, single or co-infections of Sodalis and Rickettsia, we found a high vertical transmission rate for both endosymbionts (>89%). However, we were only able to estimate these numbers for co-infected lacewings. Sodalis negatively affected the reproductive success in single and co-infected Ch. carnea, while Rickettsia showed no effect. We hypothesize that the fitness costs accrued by Sodalis infections might be more tolerable in the laboratory than in natural populations, as the latter are also prone to fluctuating environmental conditions and natural enemies. The economic and ecological importance of lacewings in biological pest control warrants a more profound understanding of its biology, which might be influenced by symbionts.

The evolution of the axonal transport toolkit.

Neurons are highly polarized cells that critically depend on long-range, bidirectional transport between the cell body and synapse for their function. This continual and highly coordinated trafficking process, which takes place via the axon, has fascinated researchers since the early 20th century. Ramon y Cajal first proposed the existence of axonal trafficking of biological material after observing that dissociation of the axon from the cell body led to neuronal degeneration. Since these first indirect observations, the field has come a long way in its understanding of this fundamental process. However, these advances in our knowledge have been aided by breakthroughs in other scientific disciplines, as well as the parallel development of novel tools, techniques and model systems. In this review, we summarize the evolution of tools used to study axonal transport and discuss how their deployment has refined our understanding of this process. We also highlight innovative tools currently being developed and how their addition to the available axonal transport toolkit might help to address key outstanding questions.

Omentin-1 and NAMPT serum concentrations are higher and CK-18 levels are lower in children and adolescents with type 1 diabetes when compared to healthy age, sex and BMI matched controls.

Background Adipokines were shown to affect glucose homeostasis and ß-cell function in patients with pancreatic dysfunction which is associated with changes in the adipose tissue secretory profile. However, information about adipokines associated with ß-cell dysfunction is lacking in pediatric patients with type 1 diabetes. Methods (1) We compared serum concentrations of nicotinamide phosphoribosyltransferase (NAMPT), omentin-1 and caspase-cleaved cytokeratin 18 fragment M30 (CK-18) in pediatric type 1 diabetes patients (n=245) and healthy age, sex and body mass index standard deviation score (BMI-SDS) matched controls (n=243). (2) We investigated the influence of insulin treatment on serum concentrations of NAMPT, omentin-1 and CK-18 in groups of patients with type 1 diabetes stratified according to the duration of their disease: at onset (n=50), ≥6 months and <5 years (n=185), ≥5 and <10 years (n=98), and ≥10 years (n=52). Results Patients at onset compared with healthy controls demonstrated no significant differences in NAMPT levels (p=0.129), whereas omentin-1 levels were elevated (p<0.001) and CK-18 levels were lowered (p=0.034). In contrast, NAMPT and omentin-1 were elevated and CK-18 serum levels were lower in longstanding patients compared to healthy controls (p<0.001). NAMPT serum levels did not change significantly during the duration of type 1 diabetes (p=0.546). At onset, omentin-1 and CK-18 levels were higher than in any group of longstanding type 1 diabetes (p<0.025). Conclusions Altered serum levels of NAMPT, omentin-1 and CK-18 in pediatric type 1 diabetes patients indicate metabolic changes caused by adipose tissue dysregulation which do not normalize during insulin therapy.

Inhibition of NAMPT sensitizes MOLT4 leukemia cells for etoposide treatment through the SIRT2-p53 pathway.

NAMPT (Nicotinamide phosphoribosyltransferase) catalyses the rate-limiting step in the NAD biosynthesis from nicotinamide and thereby regulates the activity of NAD-dependent enzymes. Cancer cells are highly dependent on NAD for energy and DNA repair processes and are assumed to be more susceptible to an inhibition of NAD synthesis than non-transformed cells. We aimed to investigate whether or not inhibition of NAMPT with its specific inhibitor FK866 can sensitize leukemia cells for chemotherapeutic agents. NAMPT protein abundance, enzymatic activity and NAD concentrations were significantly higher in Jurkat and Molt-4 leukemia cell lines compared to normal peripheral blood mononuclear cells. Combination of etoposide and FK866 caused increased cell death in leukemia cell lines compared to etoposide alone. Etoposide decreased protein abundance of NAD-dependent deacetylases SIRTUIN1. After combining etoposide and FK866 treatment SIRTUIN2 was further decreased and accumulation and acetylation of the downstream target p53 was further enhanced in MOLT4 cells. Concomitantly, protein abundance of p21 and cleaved BAX was increased. Targeting NAMPT could be a novel therapeutic strategy to enhance the efficacy of chemotherapeutic agents such as etoposide against leukemia.

Simvastatin induces apoptosis in PTEN­haploinsufficient lipoma cells.

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E­binding protein (4E­BP)­1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator­activated receptor (PPAR)γ expression. The small interfering RNA­mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Comparative analyses of glycerotoxin expression unveil a novel structural organization of the bloodworm venom system.

BACKGROUND: We present the first molecular characterization of glycerotoxin (GLTx), a potent neurotoxin found in the venom of the bloodworm Glycera tridactyla (Glyceridae, Annelida). Within the animal kingdom, GLTx shows a unique mode of action as it can specifically up-regulate the activity of Cav2.2 channels (N-type) in a reversible manner. The lack of sequence information has so far hampered a detailed understanding of its mode of action. RESULTS: Our analyses reveal three ~3.8 kb GLTx full-length transcripts, show that GLTx represents a multigene family, and suggest it functions as a dimer. An integrative approach using transcriptomics, quantitative real-time PCR, in situ hybridization, and immunocytochemistry shows that GLTx is highly expressed exclusively in four pharyngeal lobes, a previously unrecognized part of the venom apparatus. CONCLUSIONS: Our results overturn a century old textbook view on the glycerid venom system, suggesting that it is anatomically and functionally much more complex than previously thought. The herein presented GLTx sequence information constitutes an important step towards the establishment of GLTx as a versatile tool to understand the mechanism of synaptic function, as well as the mode of action of this novel neurotoxin.

Syllidae mitochondrial gene order is unusually variable for Annelida.

Complete mitochondrial genomes of five syllids (Streptosyllis sp., Eusyllis blomstrandi, Myrianida brachycephala, Typosyllis antoni and Typosyllis sp.) have been obtained using Illumina sequencing. Together with two previous studied taxa (Ramisyllis multicaudata and Trypanobia cryptica), the analysed sequences represent most of the main lineages within the family Syllidae (Anoplosyllinae, Eusyllinae, Autolytinae and Syllinae). The genomic features, gene order and phylogenetic relationships are examined. Unusual for annelids, syllid mitochondrial genomes are highly variable in their gene order. Considering genomic features, such as length, skewness, gene content, and codon bias, most similar to the rest of annelids are the genomes of E. blomstrandi and M. brachycephala, while Streptosyllis sp. and the analysed sylline taxa (R. multicaudata, T. cryptica, T. antoni and Typosyllis sp.) are the most dissimilar. Two methionine tRNA's (trnM) have been found in T. antoni and Typosyllis sp. The mt genomes of these latter taxa are the longest with numerous non-coding regions. The 13 protein coding genes, as well as the rRNA's are used to perform phylogenetic analyses that recovered the relationships within the family explored before by previous authors. The gene order in Syllidae shows very different patterns. E. blomstrandi and M. prolifera show a similar pattern to the one found in Pleistoannelida; however this might have changed at least twice within Syllidae: in Streptosyllis sp. and within Syllinae. All analysed Syllinae show different gene orders, thereby illustrating more variability as all other pleistoannelids analysed so far. The information provided herein allows a more accurate reconstruction of the possible evolutionary scenarios in Syllidae.

The Utility of Genome Skimming for Phylogenomic Analyses as Demonstrated for Glycerid Relationships (Annelida, Glyceridae).

Glyceridae (Annelida) are a group of venomous annelids distributed worldwide from intertidal to abyssal depths. To trace the evolutionary history and complexity of glycerid venom cocktails, a solid backbone phylogeny of this group is essential. We therefore aimed to reconstruct the phylogenetic relationships of these annelids using Illumina sequencing technology. We constructed whole-genome shotgun libraries for 19 glycerid specimens and 1 outgroup species (Glycinde armigera). The chosen target genes comprise 13 mitochondrial proteins, 2 ribosomal mitochondrial genes, and 4 nuclear loci (18SrRNA, 28SrRNA, ITS1, and ITS2). Based on partitioned maximum likelihood as well as Bayesian analyses of the resulting supermatrix, we were finally able to resolve a robust glycerid phylogeny and identified three clades comprising the majority of taxa. Furthermore, we detected group II introns inside the cox1 gene of two analyzed glycerid specimens, with two different insertions in one of these species. Moreover, we generated reduced data sets comprising 10 million, 4 million, and 1 million reads from the original data sets to test the influence of the sequencing depth on assembling complete mitochondrial genomes from low coverage genome data. We estimated the coverage of mitochondrial genome sequences in each data set size by mapping the filtered Illumina reads against the respective mitochondrial contigs. By comparing the contig coverage calculated in all data set sizes, we got a hint for the scalability of our genome skimming approach. This allows estimating more precisely the number of reads that are at least necessary to reconstruct complete mitochondrial genomes in Glyceridae and probably non-model organisms in general.

Hepatic NAD salvage pathway is enhanced in mice on a high-fat diet.

Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation.

A Polychaete's powerful punch: venom gland transcriptomics of Glycera reveals a complex cocktail of toxin homologs.

Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands.

The first venomous crustacean revealed by transcriptomics and functional morphology: remipede venom glands express a unique toxin cocktail dominated by enzymes and a neurotoxin.

Animal venoms have evolved many times. Venomous species are especially common in three of the four main groups of arthropods (Chelicerata, Myriapoda, and Hexapoda), which together represent tens of thousands of species of venomous spiders, scorpions, centipedes, and hymenopterans. Surprisingly, despite their great diversity of body plans, there is no unambiguous evidence that any crustacean is venomous. We provide the first conclusive evidence that the aquatic, blind, and cave-dwelling remipede crustaceans are venomous and that venoms evolved in all four major arthropod groups. We produced a three-dimensional reconstruction of the venom delivery apparatus of the remipede Speleonectes tulumensis, showing that remipedes can inject venom in a controlled manner. A transcriptomic profile of its venom glands shows that they express a unique cocktail of transcripts coding for known venom toxins, including a diversity of enzymes and a probable paralytic neurotoxin very similar to one described from spider venom. We screened a transcriptomic library obtained from whole animals and identified a nontoxin paralog of the remipede neurotoxin that is not expressed in the venom glands. This allowed us to reconstruct its probable evolutionary origin and underlines the importance of incorporating data derived from nonvenom gland tissue to elucidate the evolution of candidate venom proteins. This first glimpse into the venom of a crustacean and primitively aquatic arthropod reveals conspicuous differences from the venoms of other predatory arthropods such as centipedes, scorpions, and spiders and contributes valuable information for ultimately disentangling the many factors shaping the biology and evolution of venoms and venomous species.

Oleate rescues INS-1E ß-cells from palmitate-induced apoptosis by preventing activation of the unfolded protein response.

BACKGROUND: Saturated free fatty acids (FFAs), such as palmitate, cause ß-cell apoptosis whereas unsaturated FFAs, e.g. oleate, are not harmful. The toxicity of palmitate could be mediated through endoplasmic reticulum (ER) stress which triggers the activation of a signal responding cascade also called unfolded protein response (UPR). We investigated whether or not palmitate induced ß-cell apoptosis through UPR activation and whether or not oleate as a monounsaturated fatty acid could counteract these effects. METHODS: INS-1E ß-cells were incubated with palmitate [0.5mM], oleate [1mM] or the combination [0.5/1mM] for 1, 6 and 24h. Viability and induction of apoptosis were measured by WST-1 assay and FITC-Annexin/PI-staining, respectively. Western blot analyses were performed for UPR specific proteins and mRNA expression of target molecules was determined by qPCR. RESULTS: Palmitate significantly decreased viability (29±8.8%) of INS-1E ß-cells compared to controls after 24h. Stimulation with oleate showed no effect on viability but the combination of oleate and palmitate improved viability compared to palmitate treated cells (55±9.3%) or controls (26±5.3%). The number of apoptotic cells was increased 2-fold after 24h incubation with palmitate compared to controls. Again, oleate showed no effect but in combination ameliorated the effect of palmitate to control level. Phosphorylation of eIF2α was increased after 6 and 24h incubation with palmitate. In contrast, oleate had no effect and in combination prevented phosphorylation of eIF2α. Increased Xbp1 splicing was visible already 6h after palmitate treatment and remained elevated at 24h. The combination with oleate abolished Xbp1 splicing. Interestingly, mRNA expression of the chaperones Bip, Pdi, Calnexin and Grp94 was not altered by FFA treatment. Only the proapoptotic transcription factor Chop was significantly enhanced by palmitate incubation. In accordance with sustained cell survival the combination as well as oleate alone, did not result in increased Chop levels compared to controls. In summary, we showed that oleate protects INS-1E ß-cells from palmitate-induced apoptosis by the suppression of ER stress which was independent of chaperone activation.

Modification of beta-lactoglobulin by microbial transglutaminase under high hydrostatic pressure: localization of reactive glutamine residues.

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.