Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans.

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.

Cellular and mitochondrial adaptation mechanisms in the colon of lactating dairy cows during hyperthermia.

Heat stress causes barrier dysfunction and inflammation of the small intestine of several species. However, less is known about the molecular and cellular mechanisms underlying the response of the bovine large intestine to hyperthermia. We aimed to identify changes in the colon of dairy cows in response to constant heat stress using a proteomic approach. Eighteen lactating Holstein dairy cows were kept under constant thermoneutral conditions (16°C and 68% relative humidity [RH]; temperature-humidity index [THI] = 60) for 6 d (period 1) with free access to feed and water. Thereafter, 6 cows were equally allocated to (1) thermoneutral condition with ad libitum feeding (TNAL; 16°C, RH = 68%, THI = 60), (2) heat stress condition (HS; 28°C, RH = 50%, THI = 76) with ad libitum feeding, or (3) pair-feeding at thermoneutrality (TNPF; 16°C, RH = 68%, THI = 60) for another 7 d (period 2). Rectal temperature, milk yield, dry matter and water intake were monitored daily. Then, cows were slaughtered and colon mucosa samples were taken for proteomic analysis. Physiological data were analyzed by ANOVA and colon proteome data were processed using DESeq2 package in R. Rectal temperature was significantly higher in HS than in TNPF and TNAL cows in period 2. Proteomic analysis revealed an enrichment of activated pathways related to colonic barrier function and inflammation, heat shock proteins, AA metabolism, reduced overall protein synthesis rate, and post-transcriptional regulation induced by heat stress. Further regulations were found for enzymes of the tricarboxylic acid cycle and components of the mitochondrial electron transport chain, presumably to reduce the generation of reactive oxygen species, maintain cellular ATP levels, and prevent apoptosis in the colon of HS cows. These results highlight the cellular, extracellular, and mitochondrial adaptations of the colon during heat stress and suggest a dysfunction of the hindgut barrier integrity potentially resulting in a "leaky" colon.

Sleep Analysis in Adult C. elegans Reveals State-Dependent Alteration of Neural and Behavioral Responses.

Sleep, a state of quiescence associated with growth and restorative processes, is conserved across species. Invertebrates including the nematode Caenorhabditis elegans exhibit sleep-like states during development, satiety, and stress. Here, we describe behavior and neural activity during sleep and awake states in adult C. elegans hermaphrodites using new microfluidic methods. We observed effects of fluid flow, oxygen, feeding, odors, and genetic perturbations on long-term sleep behavior over 12 h. We developed a closed-loop sleep detection system to automatically deliver chemical stimuli to assess sleep-dependent changes to evoked neural responses in individual animals. Sleep increased the arousal threshold to aversive stimulation, yet the associated sensory neuron and first-layer interneuron responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the premotor interneurons, rather than afferent sensory circuits. However, sleep prolonged responses in appetitive chemosensory neurons, suggesting that sleep modulates responsiveness specifically across sensory systems rather than broadly damping global circuit activity.SIGNIFICANCE STATEMENT Much is known about molecular mechanisms that facilitate sleep control. However, it is unclear how these pathways modulate neural circuit-level sensory processing or how misregulation of neural activity contributes to sleep disorders. The nematode Caenorhabditis elegans provides the ability to study neural circuitry with single-neuron resolution, and recent studies examined sleep states between developmental stages and when stressed. Here, we examine an additional form of spontaneous sleep in adult C. elegans at the behavioral and neural activity levels. Using a closed-loop system, we show that delayed behavioral responses to aversive chemical stimulation during sleep arise from sleep-dependent sensorimotor modulation localized presynaptic to the premotor circuit, rather than early sensory circuits.

Insights into the Degradation of Medium-Chain-Length Dicarboxylic Acids in Cupriavidus necator H16 Reveal ß-Oxidation Differences between Dicarboxylic Acids and Fatty Acids.

Many homologous genes encoding ß-oxidation enzymes have been found in the genome of Cupriavidus necator H16 (synonym Ralstonia eutropha H16). By proteome analysis, the degradation of adipic acid was investigated and showed differences from the degradation of hexanoic acid. During ß-oxidation of adipic acid, activation with coenzyme A (CoA) is catalyzed by the two-subunit acyl-CoA ligase encoded by B0198 and B0199. The operon is completed by B0200 encoding a thiolase catalyzing the cleavage of acetyl-CoA at the end of the ß-oxidation cycle. C. necator ΔB0198-B0200 strain showed improved growth on adipic acid. Potential substitutes are B1239 for B0198-B0199 and A0170 as well as A1445 for B0200. A deletion mutant without all three thiolases showed diminished growth. The deletion of detected acyl-CoA dehydrogenase encoded by B2555 has an altered phenotype grown with sebacic acid but not adipic acid. With hexanoic acid, acyl-CoA dehydrogenase encoded by B0087 was detected on two-dimensional (2D) gels. Both enzymes are active with adipoyl-CoA and hexanoyl-CoA as substrates, but specific activity indicates a higher activity of B2555 with adipoyl-CoA. 2D gels, growth experiments, and enzyme assays suggest the specific expression of B2555 for the degradation of dicarboxylic acids. In C. necator H16, the degradation of carboxylic acids potentially changes with an increasing chain length. Two operons involved in growth with long-chain fatty acids seem to be replaced during growth on medium-chain carboxylic acids. Only two deletion mutants showed diminished growth. Replacement of deleted genes with one of the numerous homologous is likely. IMPORTANCE The biotechnologically interesting bacterium Cupriavidus necator H16 has been thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, et al., Nat Biotechnol 24:1257-1262, 2006, https://doi.org/10.1038/nbt1244). Nevertheless, the degradation of monocarboxylic fatty acids and dicarboxylic acids has not been elucidated completely. C. necator is used to produce value-added products from affordable substrates. One of our investigations' primary targets is the biotechnological production of organic acids with different and specific chain lengths. The versatile metabolism of carboxylic acids recommends C. necator H16 as a candidate for producing value-added organic products. Therefore, the metabolism of these compounds is of interest, and, for different applications in industry, understanding such central metabolic pathways is crucial.

Bioactive lipid screening during respiratory tract infections with bacterial and viral pathogens in mice.

INTRODUCTION: Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections. OBJECTIVES: Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection. METHODS: C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining. RESULTS: We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen. CONCLUSIONS: Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.

Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo.

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.

The effect of chronic, mild heat stress on metabolic changes of nutrition and adaptations in rumen papillae of lactating dairy cows.

Global warming and accompanying high ambient temperatures reduce feed intake of dairy cows and shift the blood flow from the core of the body to the periphery. As a result, hypoxia may occur in the digestive tract accompanied by disruption of the intestinal barrier, local endotoxemia and inflammation, and altered nutrient absorption. However, whether the barrier of the rumen, like the intestine, is affected by ambient heat has not been studied so far. Lactating Holstein dairy cows were subjected to heat stress at 28°C (temperature-humidity index = 76; n = 5) with ad libitum feed intake or to thermoneutral conditions at 15°C (temperature-humidity index = 60; n = 5) and pair-feeding to heat-stressed animals for a total of 4 d. Gas exchange and feed intake behavior were measured in a respiration chamber, and rumen epithelia were taken after slaughter. Heat stress significantly reduced meal size and whole-body fat oxidation but increased meal frequency and carbohydrate oxidation. The mRNA expression of toll-like receptor 4 (TLR4) and tight junction proteins and the phosphorylation of TLR4 downstream targets (interleukin-1 receptor-associated kinase 4, stress-activated protein kinase, p38 mitogen-activated protein kinase, and nuclear factor k-B) in the rumen epithelium were not affected by heat. The proteomics approach revealed increased expression of rumen epithelium proteins involved in the AMP-activated protein kinase (AMPK) and insulin signaling pathways in heat-stressed cows. Also, proteins involved in chaperone-mediated folding of proteins were upregulated, whereas those involved in antioxidant defense system were downregulated. Further, we found evidence for increased carbohydrate phosphorylation accompanied with an increased flux of carbohydrates through the hexosamine biosynthetic pathway, providing substrates for protein glycosylation. In conclusion, the mild heat stress did not induce barrier dysfunction or inflammatory responses in the rumen epithelium of dairy cows, probably because of adaptations in feed intake behavior and defense mechanisms at the tissue level.

Silk Particle Production Based on silk/PVA Phase Separation Using a Microfabricated Co-flow Device.

Polymeric particles are ideal drug delivery systems due to their cellular uptake-relevant size. Microparticles could be developed for direct injection of drug formulations into a diseased site, such as a tumor, allowing for drug retention and slow drug exposure over time through sustained release mechanisms. Bombyx mori silk fibroin has shown promise as a biocompatible biomaterial both in research and the clinic. Silk has been previously used to make particles using an emulsion-based method with poly(vinyl alcohol) (PVA). In this study, polydimethylsiloxane-based microfluidic devices were designed, fabricated, and characterized to produce silk particles through self-association of silk when exposed to PVA. Three main variables resulted in differences in particle size and size distribution, or polydispersity index (PDI). Utilizing a co-flow microfluidic device decreased the PDI of the silk particles as compared to an emulsion-based method (0.13 versus 0.65, respectively). With a flow-focusing microfluidics device, lowering the silk flow rate from 0.80 to 0.06 mL/h resulted in a decrease in the median particle size from 6.8 to 3.0 µm and the PDI from 0.12 to 0.05, respectively. Lastly, decreasing the silk concentration from 12% to 2% resulted in a decrease in the median particle size from 5.6 to 2.8 µm and the PDI from 0.81 to 0.25, respectively. Binding and release of doxorubicin, a cytotoxic drug commonly used for cancer treatment, with the fabricated silk particles was evaluated. Doxorubicin loading in the silk particles was approximately 41 µg/mg; sustained doxorubicin release occurred over 23 days. When the cytotoxicity of the released doxorubicin was tested on KELLY neuroblastoma cells, significant cell death was observed. To demonstrate the potential for internalization of the silk particles, both KELLY and THP-1-derived macrophages were exposed to fluorescently labelled silk particles for up to 24 h. With the macrophages, internalization of the silk particles was observed. Additionally, THP-1 derived macrophages exposure to silk particles increased TNF-α secretion. Overall, this microfluidics-based approach for fabricating silk particles utilizing PVA as a means to induce phase separation and silk self-assembly is a promising approach to control particle size and size distribution. These silk particles may be utilized for a variety of biomedical applications including drug delivery to multiple cell types within a tumor microenvironment.

What parameters affect knee function in patients with untreated cartilage defects: baseline data from the German Cartilage Registry.

PURPOSE: To evaluate the factors influencing the baseline Knee Injury and Osteoarthritis Outcome Score (KOOS) in patients with knee cartilage defects and planned cartilage repair surgery and to provide baseline KOOS data from a large patient population. MATERIAL AND METHODS: Between October 2013 and April 2017, a total of 2815 patients assigned for cartilage repair surgery were included into the German Cartilage Registry (KnorpelRegister DGOU) and their data were analyzed for the present study. Multivariate regression model and ANOVA were used to detect patient- and defect-specific factors with an influence on baseline KOOS. In addition, KOOS baseline data was calculated and compared according to these parameters. RESULTS: Sex, age, body mass index (BMI), and smoking status were revealed as patient-specific factors, and defect location and the number of previous knee and cartilage operations were revealed as defect-specific factors with a significant influence on baseline KOOS. Most subscores were affected in accordance with the total KOOS. Interestingly, defect ICRS grade, defect size, and symptom duration had no significant influence. The mean baseline KOOS was 56.7 (± 17.9). Men had significantly higher mean overall KOOS (60 ± 17.3 vs. 51.8 ± 17.6, p < 0.001) than women, and patients with a BMI over 30 and smokers scored significantly lower (58.07 ± 17.67 vs. 50.32 ± 17.29, p < 0.001; 57.64 ± 17.86 vs. 53.59 ± 18.06, p < 0.001). Patients with two or more previous knee operations as well as patients with more than one previous cartilage procedure also showed significantly lower overall KOOS (57.19 ± 17.89 vs. 54.56 ± 17.58, p < 0.001; 57.68 ± 18.01 vs. 52.72 ± 17.58, p < 0.001). CONCLUSION: Several factors influencing baseline KOOS data in patients with knee cartilage defects assigned for cartilage repair surgery could be detected. Their individual influence in the multivariate linear regression model was not very strong. Baseline data according to these criteria is presented in this paper.

The Proteomic Response of Bacillus pumilus Cells to Glucose Starvation.

Since starvation for carbon sources is a common condition for bacteria in nature and it can also occur in industrial fermentation processes due to mixing zones, knowledge about the response of cells to carbon starvation is beneficial. The preferred carbon source for bacilli is glucose. The response of Bacillus pumilus cells to glucose starvation using metabolic labeling and quantitative proteomics was analyzed. Glucose starvation led to an extensive reprogramming of the protein expression pattern in B. pumilus. The amounts of proteins of the central carbon metabolic pathways (glycolysis and TCC) remained stable in starving cells. Proteins for gluconeogenesis were found in higher amounts during starvation. Furthermore, many proteins involved in acquisition and usage of alternative carbon sources were present in elevated amounts in starving cells. Enzymes for fatty acid degradation and proteases and peptidases were also found in higher abundance when cells entered stationary phase. Among the proteins found in lower amounts were many enzymes involved in amino acid and nucleotide synthesis and several NRPS and PKS proteins.

Symbiotic Interplay of Fungi, Algae, and Bacteria within the Lung Lichen Lobaria pulmonaria L. Hoffm. as Assessed by State-of-the-Art Metaproteomics.

Lichens are recognized by macroscopic structures formed by a heterotrophic fungus, the mycobiont, which hosts internal autotrophic photosynthetic algal and/or cyanobacterial partners, referred to as the photobiont. We analyzed the structure and functionality of the entire lung lichen Lobaria pulmonaria L. Hoffm. collected from two different sites by state-of-the-art metaproteomics. In addition to the green algae and the ascomycetous fungus, a lichenicolous fungus as well as a complex prokaryotic community (different from the cyanobacteria) was found, the latter dominated by methanotrophic Rhizobiales. Various partner-specific proteins could be assigned to the different lichen symbionts, for example, fungal proteins involved in vesicle transport, algal proteins functioning in photosynthesis, cyanobacterial nitrogenase and GOGAT involved in nitrogen fixation, and bacterial enzymes responsible for methanol/C1-compound metabolism as well as CO-detoxification. Structural and functional information on proteins expressed by the lichen community complemented and extended our recent symbiosis model depicting the functional multiplayer network of single holobiont partners.1 Our new metaproteome analysis strongly supports the hypothesis (i) that interactions within the self-supporting association are multifaceted and (ii) that the strategy of functional diversification within the single lichen partners may support the longevity of L. pulmonaria under certain ecological conditions.

Bacillus pumilus KatX2 confers enhanced hydrogen peroxide resistance to a Bacillus subtilis PkatA::katX2 mutant strain.

BACKGROUND: Bacillus pumilus cells exhibit a significantly higher resistance to hydrogen peroxide compared to closely related Bacilli like Bacillus subtilis. RESULTS: In this study we analyzed features of the catalase KatX2 of B. pumilus as one of the most important parts of the cellular response to hydrogen peroxide. KatX2, the vegetative catalase expressed in B. pumilus, was compared to the vegetative catalase KatA of B. subtilis. Data of our study demonstrate that B. pumilus can degrade toxic concentrations of hydrogen peroxide faster than B. subtilis. By replacing B. subtilis katA gene by katX2 we could significantly enhance its resistance to H2O2 and its potential to eliminate this toxic compound. Mutant cells showed a 1.5- to 2-fold higher survival to toxic concentrations of hydrogen peroxide compared to wild type cells. Furthermore, we found reversible but also irreversible oxidations of the KatX2 protein which, in contrast to KatA, contains several cysteine residues. CONCLUSIONS: Our study indicates that the catalase KatX2 plays a major role in the increased resistance of B. pumilus to oxidative stress caused by hydrogen peroxide. Resistance to hydrogen peroxide of other Bacilli can be enhanced by exchanging the native catalase in the cells with katX2.

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.

Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.

High-throughput imaging of neuronal activity in Caenorhabditis elegans.

Neuronal responses to sensory inputs can vary based on genotype, development, experience, or stochastic factors. Existing neuronal recording techniques examine a single animal at a time, limiting understanding of the variability and range of potential responses. To scale up neuronal recordings, we here describe a system for simultaneous wide-field imaging of neuronal calcium activity from at least 20 Caenorhabditis elegans animals under precise microfluidic chemical stimulation. This increased experimental throughput was used to perform a systematic characterization of chemosensory neuron responses to multiple odors, odor concentrations, and temporal patterns, as well as responses to pharmacological manipulation. The system allowed recordings from sensory neurons and interneurons in freely moving animals, whose neuronal responses could be correlated with behavior. Wide-field imaging provides a tool for comprehensive circuit analysis with elevated throughput in C. elegans.

Characteristics and associated factors of Klee cartilage lesions: preliminary baseline-data of more than 1000 patients from the German cartilage registry (KnorpelRegister DGOU).

INTRODUCTION: Knee cartilage lesions are very frequent in arthroscopic surgery. This multi-center-study was aimed to evaluate the distribution and possible associated factors of these pathologies in more than 1000 patients. MATERIALS AND METHODS: The German cartilage registry (KnorpelRegister DGOU) started in 2013. In this paper, we present the baseline-data (distribution of knee cartilage lesions and the demographic data) of more than 1000 cases since the registries' start-up. RESULTS: A total number of 47 centers were involved into this multicenter study. A total of 1071 patients primary were registered. Degenerative knees 629 times (61.8 %) and injured knees 302 times (29.6 %) were involved. In the remaining 89 knees (8.7 %) the genesis of cartilage lesions was unclear. Single defects were observed in 792 cases (77.6 %). Most frequently the medial femoral condyle or the patella was affected. In 78 knees (7.6 %) the main-defect was associated with a defect of the corresponding joint surface. In the remaining cases complex cartilage damages were found. CONCLUSIONS: Our results are in confirmation with other multicenter studies. But these former studies did not differentiate into traumatic and degenerative lesions. Furthermore no characteristics were given regarding to single, kissing or complex lesions. Thus this database will be a sufficient instrument for the investigation of the "natural course" of cartilage lesions, but above all about the effectiveness of different treatment options.

Cartilage repair surgery for full-thickness defects of the knee in Germany: indications and epidemiological data from the German Cartilage Registry (KnorpelRegister DGOU).

PURPOSE: Treatment of cartilage defects of the knee remains an important issue with high relevance. In October 2013 the German Cartilage Registry (KnorpelRegister DGOU) was initiated in order to study indications, epidemiology and (clinical) outcome of different cartilage repair techniques. The present evaluation of the registry baseline data was initiated to report common practices of cartilage repair surgery in Germany. MATERIALS AND METHODS: 1065 consecutive patients who underwent surgical cartilage treatment of the knee have been included (complete data sets available in 1027 cases; FU rate 96.4 %) between October 1, 2013 and June 30, 2015. Data collection was performed using a web-based RDE System. All data were provided by the attending physician at the time of arthroscopic or open surgery of the affected knee. RESULTS: In 1027 cartilage repair procedures, single defects were treated in 80 % of the cases with the majority of the defects located on the medial femoral condyle, followed by the patella. Degenerative defects grade III or IV according to ICRS were treated in 60 % of the cases and therefore were found more frequently compared to traumatic or post-traumatic lesions. Autologous chondrocyte implantation (ACI) was the most common technique followed by bone marrow stimulation (BMS) and osteochondral transplantation (OCT). While ACI was performed in defects with a mean size of 4.11 cm(2) SD SD 2.16), BMS and OCT (1.51 cm(2), SD 1.19; p < 0.01) were applied in significantly smaller defects (both p < 0.01). Independent of defect size, the ratio of ACI versus BMS applications differed between different defect locations. ACI was used preferably in defects located on the patella. CONCLUSION: The present analysis of data from the German Cartilage Registry shows that the vast majority of cartilage repair procedures were applied in degenerative, non-traumatic cartilage defects. Experts in Germany seem to follow the national and international guidelines in terms that bone marrow stimulation is applied in smaller cartilage defects while cell-based therapies are used for the treatment of larger cartilage defects. In patellar cartilage defects a trend towards the use of cell-based therapies has been observed.

CodY regulates expression of the Bacillus subtilis extracellular proteases Vpr and Mpr.

UNLABELLED: CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr- or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE: CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis, more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis, CodY can now be seen to regulate the entire protein utilization pathway.

An alternative method for serum protein depletion/enrichment by precipitation at mildly acidic pH values and low ionic strength.

Serum proteome analysis is severely hampered by the extreme dynamic range of protein concentrations, but tools for the specific depletion of highly abundant serum proteins lack for most farm and companion animals. A well-established alternative strategy to reduce the dynamic range of plasma protein concentrations, treatment with combinatorial peptide ligand libraries (CPLL), is generally applicable but requires large amounts of sample. Therefore, additional depletion/enrichment protocols for plasma and serum samples from animals are desirable. In this respect, we have tested a protein precipitate that formed after withdrawal of salt from human, bovine, or porcine serum at pH 4.2. The bovine sample was composed of over 300 proteins making it a potential source for biomarker discovery. Precipitation was highly reproducible and the concentrations of albumin and other highly abundant serum proteins were strongly reduced. In comparison to the CPLL treatment, precipitation did not introduce any selection bias based on hydrophathy or pI. However, the composition of both preparations was partially complementary. Salt withdrawal at pH 4.2 is suggested as additional depletion/enrichment strategy for serum samples. Also, we point out that the removal of precipitates from serum samples under the described conditions bears the risk of losing a valuable protein fraction.