Three-dimensional (3D) stereoscopic vision is crucial to perform any kind of manual task. The reduction from real life 3D to virtual two-dimensional (2D) sight is a major challenge in minimally invasive surgery (MIS). A 3D display technique has been shown to reduce operation time and mistakes and to improve the learning curve. Therefore, the use of a3D display technique seems to optimize surgical performance for novice and experienced surgeons. Inspired by consumer electronics, a 4K display technique was recently introduced to MIS. Due to its high resolution and zoom effect, surgeons should benefit from it. The aim of this study is to evaluate if "state-of-the-art" 3D- vs. 4K-display techniques could influence surgical performance.

This study aimed to investigate the effect factors associated with the postoperative dissatisfaction of patients undergoing open-door laminoplasty for cervical OPLL.

Flavone C-glycosides are difficult to be deglycosylated using traditional chemical methods due to their solid carbon-carbon bond between sugar moieties and aglycones; however, some bacteria may easily cleave this bond because they generate various specific enzymes.

Environmental exposures are related to the risk of some types of cancer, and children are the most vulnerable group of people. This study seeks to present the methodological approaches used in the papers of our group about risk of childhood cancers in the vicinity of pollution sources (industrial and urban sites). A population-based case-control study of incident childhood cancers in Spain and their relationship with residential proximity to industrial and urban areas was designed. Two methodological approaches using mixed multiple unconditional logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) were developed: (a) "near vs. far" analysis, where possible excess risks of cancers in children living near ("near") versus those living far ("far") from industrial and urban areas were assessed; and (b) "risk gradient" analysis, where the risk gradient in the vicinity of industries was assessed. For each one of the two approaches, three strategies of analysis were implemented: "joint", "stratified", and "individualized" analysis. Incident cases were obtained from the Spanish Registry of Childhood Cancer (between 1996 and 2011).

Arterial stiffness is closely related to the process of atherosclerosis, an independent cardiovascular risk factor, and predictive of future cardiovascular events and mortality. Recently, we showed that magnesium citrate supplementation results in a clinically relevant improvement of arterial stiffness. It remained unclear whether the observed effect was due to magnesium or citrate, and whether other magnesium compounds may have similar effects. Therefore, we aim to study the long-term effects of magnesium citrate, magnesium oxide and magnesium sulfate on arterial stiffness. In addition, we aim to investigate possible underlying mechanisms, including changes in blood pressure and changes in gut microbiota diversity.

The molecular mechanisms behind poor foetal growth are not fully known. The aim of this study was to explore global microRNA expression in placentas of infants born small for gestational age (SGA) compared to infants with a normal birth weight (NBW).

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is the only human neurotrophic factor with an evolutionarily-conserved C. elegans homolog, Y54G2A.23 or manf-1. MANF is a small, soluble, endoplasmic-reticulum (ER)-resident protein that is secreted upon ER stress and promotes survival of target cells such as neurons. However, the role of MANF in ER stress and its mechanism of cellular protection are not clear and the function of MANF in C. elegans is only beginning to emerge. In this study, we show that depletion of C. elegans manf-1 causes a slight decrease in lifespan and brood size; furthermore, combined depletion of manf-1 and the IRE-1/XBP-1 ER stress/UPR pathway resulted in sterile animals that did not produce viable progeny. We demonstrate upregulation of markers of ER stress in L1 larval nematodes, as measured by hsp-3 and hsp-4 transcription, upon depletion of manf-1 by RNAi or mutation; however, there was no difference in tunicamycin-induced expression of hsp-3 and hsp-4 between wild-type and MANF-deficient worms. Surprisingly, larval growth arrest observed in wild-type nematodes reared on tunicamycin is completely prevented in the manf-1 (tm3603) mutant. Transcriptional microarray analysis revealed that manf-1 mutant L1 larvae exhibit a novel modulation of innate immunity genes in response to tunicamycin. The hypothesis that manf-1 negatively regulates the innate immunity pathway is supported by our finding that the development of manf-1 mutant larvae compared to wild-type larvae is not inhibited by growth on P. aeruginosa. Together, our data represent the first characterization of C. elegans MANF as a key modulator of organismal ER stress and immunity.

ASN100 is a novel antibody combination of two fully human IgG1(κ) monoclonal antibodies (MAbs), ASN-1 and ASN-2, which neutralize six Staphylococcus aureus cytotoxins, alpha-hemolysin (Hla) and five bicomponent leukocidins. We assessed the safety, tolerability, and serum and lung pharmacokinetics of ASN100 in a randomized, double-blind, placebo-controlled single-dose-escalation first-in-human study. Fifty-two healthy volunteers were enrolled and randomized to receive either ASN-1, ASN-2, a combination of both MAbs (ASN100), or a corresponding placebo. Thirty-two subjects in the double-blind dose escalation portion of the study received ASN-1 or ASN-2 at a 200-, 600-, 1,800-, or 4,000-mg dose, or placebo. Eight subjects received both MAbs simultaneously in a 1:1 ratio (ASN100) at 3,600 or 8,000 mg, or they received placebos. Twelve additional subjects received open-label ASN100 at 3,600 or 8,000 mg to assess the pharmacokinetics of ASN-1 and ASN-2 in epithelial lining fluid (ELF) by bronchoalveolar lavage fluid sampling. Subjects were monitored for 98 days (double-blind cohorts) or 30 days (open-label cohorts) for safety assessment. No dose-limiting toxicities were observed, and all adverse events were mild and transient, with only two adverse events considered possibly related to the investigational product. ASN100 exhibited linear serum pharmacokinetics with a half-life of approximately 3 weeks and showed detectable penetration into the ELF. No treatment-emergent anti-drug antibody responses were detected. The toxin neutralizing potency of ASN100 in human serum was confirmed up to 58 days postdosing. The favorable safety profile, ELF penetration, and maintained functional activity in serum supported the further clinical development of ASN100.

Extramedical use of, and associated harms with pharmaceutical opioids are common. Analysis of coded ambulance clinical records provides a unique opportunity to examine a national population-level indicator of relative harms. This protocol describes an observational study with three aims: (1) to compare supply adjusted rates of pharmaceutical opioid-related ambulance attendances for buprenorphine, codeine, fentanyl, oxycodone, oxycodone-naloxone, morphine, pethidine, tramadol and tapentadol; (2) to compare presentation characteristics for these commonly used pharmaceutical opioids and (3) to describe the context surrounding ambulance presentations related to oxycodone, a widely used opioid with an established abuse liability, and tapentadol, a more recent 'atypical' opioid on the Australian market, with fewer studies that have directly examined signals of extramedical use.

The endocannabinoid (eCB) system regulates energy homeostasis and is linked to obesity development. However, the exact dynamic and regulation of eCBs in the hypothalamus during obesity progression remain incompletely described and understood. Our study examined the time course of responses in two hypothalamic eCBs, 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamine (AEA), in male and female mice during diet-induced obesity and explored the association of eCB levels with changes in brown adipose tissue (BAT) thermogenesis and body weight. We fed mice a high-fat diet (HFD), which induced a transient increase (substantial at 7 days) in hypothalamic eCBs, followed by a progressive decrease to basal levels with a long-term HFD. This transient rise at early stages of obesity is considered a physiologic compensatory response to BAT thermogenesis, which is activated by diet surplus. The eCB dynamic was sexually dimorphic: hypothalamic eCBs levels were higher in female mice, who became obese at later time points than males. The hypothalamic eCBs time course positively correlated with thermogenesis activation, but negatively matched body weight, leptinemia, and circulating eCB levels. Increased expression of eCB-synthetizing enzymes accompanied the transient hypothalamic eCB elevation. Icv injection of eCB did not promote BAT thermogenesis; however, administration of thermogenic molecules, such as central leptin or a peripheral β3-adrenoreceptor agonist, induced a significant increase in hypothalamic eCBs, suggesting a directional link from BAT thermogenesis to hypothalamic eCBs. This study contributes to the understanding of hypothalamic regulation of obesity.

The actomyosin network is involved in crucial cellular processes including morphogenesis, cell adhesion, apoptosis, proliferation, differentiation, and collective cell migration in Drosophila, Caenorhabditiselegans, and mammals. Here, we demonstrate that Drosophila larval blood stem-like progenitors require actomyosin activity for their maintenance. Genetic loss of the actomyosin network from progenitors caused a decline in their number. Likewise, the progenitor population increased upon sustained actomyosin activation via phosphorylation by Rho-associated kinase. We show that actomyosin positively regulates larval blood progenitors by controlling the maintenance factor Cubitus interruptus (Ci). Overexpression of the maintenance signal via a constitutively activated construct (ci.HA) failed to sustain Ci-155 in the absence of actomyosin components like Zipper (zip) and Squash (sqh), thus favoring protein kinase A (PKA)-independent regulation of Ci activity. Furthermore, we demonstrate that a change in cortical actomyosin assembly mediated by DE-cadherin modulates Ci activity, thereby determining progenitor status. Thus, loss of cell adhesion and downstream actomyosin activity results in desensitization of the progenitors to Hh signaling, leading to their differentiation. Our data reveal how cell adhesion and the actomyosin network cooperate to influence patterning, morphogenesis, and maintenance of the hematopoietic stem-like progenitor pool in the developing Drosophila hematopoietic organ.

Clostridium difficile disease is mediated primarily by toxins A and B (TcdA and TcdB, respectively). The receptor binding domains (RBD) of TcdA and TcdB are immunogenic, and anti-RBD antibodies are protective. Since these toxins act locally, an optimal C. difficile vaccine would generate both systemic and mucosal responses. We have repurposed an attenuated Salmonella enterica serovar Typhimurium strain (YS1646) to produce such a vaccine. Plasmid-based candidates expressing either the TcdA or TcdB RBD were screened. Different vaccine routes and schedules were tested to achieve detectable serum and mucosal antibody titers in C57BL/6J mice. When given in a multimodality schedule over 1 week (intramuscularly and orally [p.o.] on day 0 and p.o. on days 2 and 4), several candidates provided 100% protection against lethal challenge. Substantial protection (82%) was achieved with combined p.o. TcdA and TcdB vaccination alone (days 0, 2, and 4). These data demonstrate the potential of the YS1646-based vaccines for C. difficile and strongly support their further development.

In most eukaryotes, telomerase counteracts chromosome erosion by adding repetitive sequence to terminal ends. Drosophila melanogaster instead relies on specialized retrotransposons that insert exclusively at telomeres. This exchange of goods between host and mobile element-wherein the mobile element provides an essential genome service and the host provides a hospitable niche for mobile element propagation-has been called a "genomic symbiosis." However, these telomere-specialized, jockey family retrotransposons may actually evolve to "selfishly" overreplicate in the genomes that they ostensibly serve. Under this model, we expect rapid diversification of telomere-specialized retrotransposon lineages and, possibly, the breakdown of this ostensibly symbiotic relationship. Here we report data consistent with both predictions. Searching the raw reads of the 15-Myr-old melanogaster species group, we generated de novo jockey retrotransposon consensus sequences and used phylogenetic tree-building to delineate four distinct telomere-associated lineages. Recurrent gains, losses, and replacements account for this retrotransposon lineage diversity. In Drosophila biarmipes, telomere-specialized elements have disappeared completely. De novo assembly of long reads and cytogenetics confirmed this species-specific collapse of retrotransposon-dependent telomere elongation. Instead, telomere-restricted satellite DNA and DNA transposon fragments occupy its terminal ends. We infer that D. biarmipes relies instead on a recombination-based mechanism conserved from yeast to flies to humans. Telomeric retrotransposon diversification and disappearance suggest that persistently "selfish" machinery shapes telomere elongation across Drosophila rather than completely domesticated, symbiotic mobile elements.

Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.

Cell migration is essential to embryonic development, wound healing, and cancer cell dissemination. Cells move via leading-edge protrusion, substrate adhesion, and retraction of the cell's rear. The molecular mechanisms by which extracellular cues signal to the actomyosin cytoskeleton to control these motility mechanics are poorly understood. The growth factor-responsive and oncogenically activated protein extracellular signal-regulated kinase (ERK) promotes motility by signaling in actin polymerization-mediated edge protrusion. Using a combination of immunoblotting, co-immunoprecipitation, and myosin-binding experiments and cell migration assays, we show here that ERK also signals to the contractile machinery through its substrate, p90 ribosomal S6 kinase (RSK). We probed the signaling and migration dynamics of multiple mammalian cell lines and found that RSK phosphorylates myosin phosphatase-targeting subunit 1 (MYPT1) at Ser-507, which promotes an interaction of Rho kinase (ROCK) with MYPT1 and inhibits myosin targeting. We find that by inhibiting the myosin phosphatase, ERK and RSK promote myosin II-mediated tension for lamella expansion and optimal edge dynamics for cell migration. These findings suggest that ERK activity can coordinately amplify both protrusive and contractile forces for optimal cell motility.

Long-term consumption of dietary fiber is generally considered beneficial for weight management and metabolic health, but the results of interventions vary greatly depending on the type of dietary fibers involved. This study provides a comprehensive evaluation of the effects of a specific dietary fiber consisting of a wheat-bran extract enriched in arabinoxylan-oligosaccharides (AXOS) in a human intervention trial. An integrated multi-omics analysis has been carried out to evaluate the effects of an intervention trial with an AXOS-enriched diet in overweight individuals with indices of metabolic syndrome. Microbiome analyses were performed by shotgun DNA sequencing in feces; in-depth metabolomics using nuclear magnetic resonance in fecal, urine, and plasma samples; and massive lipid profiling using mass spectrometry in fecal and serum/plasma samples. In addition to their bifidogenic effect, we observed that AXOS boost the proportion of Prevotella species. Metagenome analysis showed increases in the presence of bacterial genes involved in vitamin/cofactor production, glycan metabolism, and neurotransmitter biosynthesis as a result of AXOS intake. Furthermore, lipidomics analysis revealed reductions in plasma ceramide levels. Finally, we observed associations between Prevotella abundance and short-chain fatty acids (SCFAs) and succinate concentration in feces and identified a potential protective role of Eubacterium rectale against metabolic disease given that its abundance was positively associated with plasma phosphatidylcholine levels, thus hypothetically reducing bioavailability of choline for methylamine biosynthesis. The metagenomics, lipidomics, and metabolomics data integration indicates that sustained consumption of AXOS orchestrates a wide variety of changes in the gut microbiome and the host metabolism that collectively would impact on glucose homeostasis. (This study has been registered at ClinicalTrials.gov under identifier NCT02215343)IMPORTANCE The use of dietary fiber food supplementation as a strategy to reduce the burden of diet-related diseases is a matter of study given its cost-effectiveness and the positive results demonstrated in clinical trials. This multi-omics assessment, on different biological samples of overweight subjects with signs of metabolic syndrome, sheds light on the early and less evident effects of short-term AXOS intake on intestinal microbiota and host metabolism. We observed a deep influence of AXOS on gut microbiota beyond their recognized bifidogenic effect by boosting concomitantly a wide diversity of butyrate producers and Prevotella copri, a microbial species abundant in non-Westernized populations with traditional lifestyle and diets enriched in fresh unprocessed foods. A comprehensive evaluation of hundreds of metabolites unveiled new benefits of the AXOS intake, such as reducing the plasma ceramide levels. Globally, we observed that multiple effects of AXOS consumption seem to converge in reversing the glucose homeostasis impairment.

Before the invention of electric lighting, humans were primarily exposed to intense (>300 lux) or dim (<30 lux) environmental light-stimuli at extreme ends of the circadian system's dose-response curve to light. Today, humans spend hours per day exposed to intermediate light intensities (30-300 lux), particularly in the evening. Interindividual differences in sensitivity to evening light in this intensity range could therefore represent a source of vulnerability to circadian disruption by modern lighting. We characterized individual-level dose-response curves to light-induced melatonin suppression using a within-subjects protocol. Fifty-five participants (aged 18-30) were exposed to a dim control (<1 lux) and a range of experimental light levels (10-2,000 lux for 5 h) in the evening. Melatonin suppression was determined for each light level, and the effective dose for 50% suppression (ED50) was computed at individual and group levels. The group-level fitted ED50 was 24.60 lux, indicating that the circadian system is highly sensitive to evening light at typical indoor levels. Light intensities of 10, 30, and 50 lux resulted in later apparent melatonin onsets by 22, 77, and 109 min, respectively. Individual-level ED50 values ranged by over an order of magnitude (6 lux in the most sensitive individual, 350 lux in the least sensitive individual), with a 26% coefficient of variation. These findings demonstrate that the same evening-light environment is registered by the circadian system very differently between individuals. This interindividual variability may be an important factor for determining the circadian clock's role in human health and disease.

Cerebral malaria is a severe neurological complication associated with sequestration of Plasmodium falciparum-infected erythrocytes (IE) in the brain microvasculature, but the specific binding interactions remain under debate. Here, we have generated an engineered three-dimensional (3D) human brain endothelial microvessel model and studied P. falciparum binding under the large range of physiological flow velocities that occur in both health and disease. Perfusion assays on 3D microvessels reveal previously unappreciated phenotypic heterogeneity in parasite binding to tumor necrosis factor alpha (TNF-α)-activated brain endothelial cells. While clonal parasite lines expressing a group B P. falciparum erythrocyte membrane protein 1 (PfEMP1) present an increase in binding to activated 3D microvessels, P. falciparum-IE expressing DC8-PfEMP1 present a decrease in binding. The differential response to endothelium activation is mediated by surface expression changes of endothelial protein C receptor (EPCR) and intercellular adhesion molecule 1 (ICAM-1). These findings demonstrate heterogeneity in parasite binding and provide evidence for a parasite strategy to adapt to a changing microvascular environment during infection. The engineered 3D human brain microvessel model provides new mechanistic insight into parasite binding and opens opportunities for further studies on malaria pathogenesis and parasite-vessel interactions.IMPORTANCE Cerebral malaria research has been hindered by the inaccessibility of the brain. Here, we have developed an engineered 3D human brain microvessel model that mimics the blood flow rates and architecture of small blood vessels to study how P. falciparum-infected human erythrocytes attach to brain endothelial cells. By studying parasite lines with different adhesive properties, we show that the malaria parasite binding rate is heterogeneous and strongly influenced by physiological differences in flow and whether the endothelium has been previously activated by TNF-α, a proinflammatory cytokine that is linked to malaria disease severity. We also show the importance of human EPCR and ICAM-1 in parasite binding. Our model sheds new light on how P. falciparum binds within brain microvessels and provides a powerful method for future investigations of recruitment of human brain pathogens to the blood vessel lining of the brain.

Efficient live-imaging methods are pivotal to understand fungal morphogenesis, especially as it relates to interactions with host immune cells and mechanisms of antifungal drugs. Due to the notable similarities in growth patterns of neuronal cells and mycelial networks, we sought to repurpose the NeuroTrack (NT) processing module of the IncuCyte time-lapse microscopy system as a tool to quantify mycelial growth and branching of pathogenic fungi. We showed the robustness of NT analysis to study Candida albicans and five different molds and confirmed established characteristics of mycelial growth kinetics. We also documented high intra- and interassay reproducibility of the NT module for a spectrum of spore inocula and culture periods. Using GFP-expressing Aspergillus fumigatus and Rhizopus arrhizus, the feasibility of fluorescence-based NT analysis was validated. In addition, we performed proof-of-concept experiments of NT analysis for several translational applications such as studying the morphogenesis of a filamentation-defective C. albicans mutant, the effects of different classes of antifungals (polyenes, azoles, and echinocandins), and coculture with host immune cells. High accuracy was found, even at high immune cell-to-fungus ratios or in the presence of fungal debris. For antifungal efficacy studies, addition of a cytotoxicity dye further refined IncuCyte-based analysis, facilitating real-time determination of fungistatic and fungicidal activity in a single assay. Complementing conventional MIC-based assays, NT analysis is an appealing method to study fungal morphogenesis and viability in the context of antifungal compound screening and evaluation of novel immune therapeutics.IMPORTANCE Pathogenic fungi remain a major cause of infectious complications in immunocompromised patients. Microscopic techniques are crucial for our understanding of fungal biology, host-pathogen interaction, and the pleiotropic effects of antifungal drugs on fungal cell growth and morphogenesis. Taking advantage of the morphological similarities of neuronal cell networks and mycelial growth patterns, we employed the IncuCyte time-lapse microscopy system and its NeuroTrack image analysis software package to study growth and branching of a variety of pathogenic yeasts and molds. Using optimized image processing definitions, we validated IncuCyte NeuroTrack analysis as a reliable and efficient tool for translational applications such as antifungal efficacy evaluation and coculture with host immune effector cells. Hence, the IncuCyte system and its NeuroTrack module provide an appealing platform for efficient in vitro studies of antifungal compounds and immunotherapeutic strategies in medical mycology.

Bacterial antibiotic resistance modulation by small signaling molecules is an emerging mechanism that has been increasingly reported in recent years. Several studies indicate that indole, an interkingdom signaling molecule, increases bacterial antibiotic resistance. However, the mechanism through which indole reduces antibiotic resistance is largely unknown. In this study, we demonstrated a novel mechanism for indole-mediated reversal of intrinsic antibiotic resistance in Lysobacter This reversal was facilitated by a novel BtuD-associated dual-function importer that can transfer both vitamin B12 and antibiotics. Indole stimulated btuD overexpression and promoted efficient absorption of extracellular vitamin B12; meanwhile, the weak selectivity of the importer caused cells to take up excessive doses of antibiotics that resulted in cell death. Consistently, btuD deletion and G48Y/K49D substitution led to marked reductions in the uptake of both antibiotics and vitamin B12 This novel mechanism is common across multiple bacterial species, among which the Q-loop amino acid of BtuD proteins is Glu (E) instead of Gln (Q). Interestingly, the antibiotic resistance of Lysobacter spp. can be restored by another small quorum sensing signaling factor, 13-methyltetradecanoic acid, designated LeDSF, in response to bacterial population density. This work highlights the mechanisms underlying dynamic regulation of bacterial antibiotic resistance by small signaling molecules and suggests that the effectiveness of traditional antibiotics could be increased by coupling them with appropriate signaling molecules.IMPORTANCE Recently, signaling molecules were found to play a role in mediating antibiotic resistance. In this study, we demonstrated that indole reversed the intrinsic antibiotic resistance (IRAR) of multiple bacterial species by promoting the expression of a novel dual-function importer. In addition, population-dependent behavior induced by 13-methyltetradecanoic acid, a quorum sensing signal molecule designated LeDSF, was involved in the IRAR process. This study highlights the dynamic regulation of bacterial antibiotic resistance by small signaling molecules and provides direction for new therapeutic strategies using traditional antibiotics in combination with signaling molecules.