Feeding characteristics of healthy infants without reported feeding impairments throughout the first month of life.

OBJECTIVE: Elucidate characteristics of feeding performance in healthy infants without reported feeding problems throughout the first month of life. STUDY DESIGN: Feeding was monitored in 61 healthy infants by caregiver report for 48 h a week from birth to 4 weeks old. Outcomes included feeding modality, how much they consumed, how long the feed lasted, and how many coughing episodes the infant exhibited. Data were analyzed with descriptive and non-parametric statistics. RESULT: The majority of infants (68%) exhibited at least one problematic feeding behavior. Infants consumed 68 ml/feed over 20 min, though the milk volumes and feed durations were highly variable. Coughing occurred an average of 2 feeds per day. No significant change in coughing was observed throughout the first month of life (p = 0.64). Infants coughed significantly less during breast feeds than bottle feeds (p = 0.02). CONCLUSION: Healthy term infants exhibit what appear to be normal developmental imperfections in feeding performance throughout the first month of life.

Chemical and toxicological assessment of leachates from UV-degraded plastic materials using in-vitro bioassays.

The broad use of plastics and the persistence of the material results in plastic residues being found practically everywhere in the environment. If plastics remain in the (aquatic) environment, natural weathering leads to degradation processes and compounds may leach from plastic into the environment. To investigate the impact of degradation process on toxicity of leachates, different types of UV irradiation (UV-C, UV-A/B) were used to simulate weathering processes of different plastic material containing virgin as well as recyclate material and biodegradable polymers. The leached substances were investigated toxicologically using in-vitro bioassays. Cytotoxicity was determined by the MTT-assay, genotoxicity by using the p53-CALUX and Umu-assay, and estrogenic effects by the ERα-CALUX. Genotoxic as well as estrogenic effects were detected in different samples depending on the material and the irradiation type. In four leachates of 12 plastic species estrogenic effects were detected above the recommended safety level of 0.4 ng 17ß-estradiol equivalents/L for surface water samples. In the p53-CALUX and in the Umu-assay leachates from three and two, respectively, of 12 plastic species were found to be genotoxic. The results of the chemical analysis show that plastic material releases a variety of known and unknown substances especially under UV radiation, leading to a complex mixture with potentially harmful effects. In order to investigate these aspects further and to be able to give recommendations for the use of additives in plastics, further effect-related investigations are advisable.

YgfB increases ß-lactam resistance in Pseudomonas aeruginosa by counteracting AlpA-mediated ampDh3 expression.

YgfB-mediated ß-lactam resistance was recently identified in multi drug resistant Pseudomonas aeruginosa. We show that YgfB upregulates expression of the ß-lactamase AmpC by repressing the function of the regulator of the programmed cell death pathway AlpA. In response to DNA damage, the antiterminator AlpA induces expression of the alpBCDE autolysis genes and of the peptidoglycan amidase AmpDh3. YgfB interacts with AlpA and represses the ampDh3 expression. Thus, YgfB indirectly prevents AmpDh3 from reducing the levels of cell wall-derived 1,6-anhydro-N-acetylmuramyl-peptides, required to induce the transcriptional activator AmpR in promoting the ampC expression and ß-lactam resistance. Ciprofloxacin-mediated DNA damage induces AlpA-dependent production of AmpDh3 as previously shown, which should reduce ß-lactam resistance. YgfB, however, counteracts the ß-lactam enhancing activity of ciprofloxacin by repressing ampDh3 expression and lowering the benefits of this drug combination. Altogether, YgfB represents an additional player in the complex regulatory network of AmpC regulation.

High-throughput automated methods for classical and operant conditioning of Drosophila larvae.

Learning which stimuli (classical conditioning) or which actions (operant conditioning) predict rewards or punishments can improve chances of survival. However, the circuit mechanisms that underlie distinct types of associative learning are still not fully understood. Automated, high-throughput paradigms for studying different types of associative learning, combined with manipulation of specific neurons in freely behaving animals, can help advance this field. The Drosophila melanogaster larva is a tractable model system for studying the circuit basis of behaviour, but many forms of associative learning have not yet been demonstrated in this animal. Here, we developed a high-throughput (i.e. multi-larva) training system that combines real-time behaviour detection of freely moving larvae with targeted opto- and thermogenetic stimulation of tracked animals. Both stimuli are controlled in either open- or closed-loop, and delivered with high temporal and spatial precision. Using this tracker, we show for the first time that Drosophila larvae can perform classical conditioning with no overlap between sensory stimuli (i.e. trace conditioning). We also demonstrate that larvae are capable of operant conditioning by inducing a bend direction preference through optogenetic activation of reward-encoding serotonergic neurons. Our results extend the known associative learning capacities of Drosophila larvae. Our automated training rig will facilitate the study of many different forms of associative learning and the identification of the neural circuits that underpin them.

Locomotor behavior of Neocaridina palmata: a study with leachates from UV-weathered microplastics.

Weathering of plastics leads to the formation of increasingly smaller particles with the release of chemical compounds. The latter occurs with currently unknown environmental impacts. Leachate-induced effects of weathered microplastics (MPs) are therefore of increasing concern. To investigate the toxicity of the chemical mixtures from such plastics, we exposed the freshwater shrimp Neocaridina palmata to enriched leachates from unweathered and artificially weathered (UV-A/B light) MPs (≤1 mm) from recycled low-density polyethylene (LDPE-R) pellets and from a biodegradable, not fully bio-based starch blend (SB) foil. We analyzed the individual locomotor activity (moved distance and frozen events) on day 1, 3, 7 and 14 of exposure to five leachate concentrations equivalent to 0.40-15.6 g MPs L-1, representing the upper scale of MPs that have been found in the environment. The median moved distance did not change as a function of concentration, except for the unweathered SB treatment on day 14 that indicated hyperactivity with increasing concentrations. Significant impacts were solely detected for few concentrations and exposure days. Generally, no consistent trend was observed across the experiments. We further assessed the baseline toxicity of the samples in the Microtox assay and detected high bioluminescence inhibitions of the bacterium Aliivibrio fischeri. This study demonstrates that neither the recycled nor the biodegradable material are without impacts on test parameters and therefore cannot be seen as safe alternative for conventional plastics regarding the toxicity. However, the observed in vitro toxicity did not result in substantial effects on the behavior of shrimps. Overall, we assume that the two endpoints examined in the atyid shrimp N. palmata were not sensitive to chemicals leaching from plastics or that effects on the in vivo level affect other toxic endpoints which were not considered in this study.

Particle shape does not affect ingestion and egestion of microplastics by the freshwater shrimp Neocaridina palmata.

The ingestion of microplastics (MPs) is well documented for various animals and spherical MPs (beads) in many studies. However, the retention time and egestion of MPs have been examined less, especially for irregular MPs (fragments) which are predominantly found in the environment. Furthermore, the accumulation of such particles in the gastrointestinal tract is likely to determine whether adverse effects are induced. To address this, we investigated if the ingestion and egestion of beads are different to those of fragments in the freshwater shrimp Neocaridina palmata. Therefore, organisms were exposed to 20-20,000 particles L-1 of either polyethylene (PE) beads (41 µm and 87 µm) or polyvinyl chloride (PVC) fragments (<63 µm). Moreover, shrimps were exposed to 20,000 particles L-1 of either 41 µm PE and 11 µm polystyrene (PS) beads or the PVC fragments for 24 h, followed by a post-exposure period of 4 h to analyze the excretion of particles. To simulate natural conditions, an additional fragment ingestion study was performed in the presence of food. After each treatment, the shrimps were analyzed for retained or excreted particles. Our results demonstrate that the ingestion of beads and fragments were concentration-dependent. Shrimps egested 59% of beads and 18% of fragments within 4 h. Particle shape did not significantly affect MP ingestion or egestion, but size was a relevant factor. Medium- and small-sized beads were frequently ingested. Furthermore, fragment uptake decreased slightly when co-exposed to food, but was not significantly different to the treatments without food. Finally, the investigations highlight that the assessment of ingestion and egestion rates can help to clarify whether MPs remain in specific organisms and, thereby, become a potential health threat.

Enhanced in vitro toxicity of plastic leachates after UV irradiation.

Plastics can release numerous chemicals and thereby, contribute to the chemical pollution in aquatic systems. To which extent environmental degradation processes influence the release of plastic chemicals, is currently unknown and subject of research. We therefore evaluated aqueous leachates of 12 differently formulated plastics (e.g., pre-production, post-industrial and recycled pellets as well as final products) using in vitro bioassays and chemical analysis via LC-HRMS nontarget approach. We weathered these plastics by UV irradiation (UV-C and UV-A/B) under laboratory conditions in dryness and a subsequent leaching period in ultrapure water ('atmospheric' weathering) or directly in water ('aquatic' weathering, UV-A/Baq). A dark control (DC) without UV light served as a reference treatment. Some plastics triggered several toxicological endpoints (low-density polyethylene recyclate (LDPE-R), starch blend (SB), bio-based polybutylene succinate (Bio-PBS) and polyvinyl chloride (PVC)), whereas others caused little to no effects (polyethylene terephthalate (PET), polystyrene (PS), polypropylene (PP) and LDPE). UV irradiation enhanced the plastics' toxicity, even for samples initially evaluated as toxicologically inconspicuous. The plastic samples caused oxidative stress (85%), baseline toxicity (42%), antiestrogenicity (40%) and antiandrogenicity (27%). Positive findings were measured after UV-C (63%) and UV-A/Baq (50%) treatments, followed by UV-A/B (48%) and DC (33%). Overall, we detected between 42 (DC) and 2896 (UV-A/Baq) chemical compounds. Our study demonstrates that differently formulated plastics leach toxic chemicals. UV exacerbates the plastics' toxicity by either generating active compounds and/or by facilitating their release. UV light even leads to the release of bioactive compounds from plastics of low chemical complexity. To prevent the exposure to plastic-associated chemicals, the application of chemicals could be reduced to a minimum, while on a regulatory level the evaluation of plastic eluates could be another focal point next to singular compounds.

Chemicals associated with biodegradable microplastic drive the toxicity to the freshwater oligochaete Lumbriculus variegatus.

Microplastics (MPs) as complex synthetic pollutants represent a growing concern for the aquatic environment. Previous studies examined the toxicity of MPs, but infrequently used a natural particle control such as kaolin. The cause of toxicity, either the physical structure of the particles or chemical components originating from the MPs, has rarely been resolved. Moreover, the ecotoxicological assessment of biodegradable plastics has received little attention. To narrow down the main driver for toxicity of irregular biodegradable MPs, we conducted a series of 28-days sediment toxicity tests with the freshwater oligochaete Lumbriculus variegatus and recorded the number of worms and dry weight as endpoints. Therefore, MPs containing several biodegradable polymers were either mixed with the sediment or layered on the sediment surface with concentrations from 1 to 8.4% sediment dw-1. Kaolin particles were evaluated in parallel as particle control. Furthermore, aqueous leachates and methanolic extracts as MP equivalents as well as solvent-treated, presumably pure MPs were investigated after mixing them into the sediment. Our results reveal that MP mixed with the sediment induced stronger adverse effects than layered MP. Kaolin particles caused no adverse effects. In contrast, they enhanced dry weight in both applications. The impact of aqueous leachates was comparable to the control without MPs, whereas methanolic extracts affected the worm number at the highest concentration with 100% mortality. Solvent-treated, presumably pure MP resulted in mostly higher worm numbers when compared to untreated MPs mixed into the sediment. This study demonstrates that MPs mixed into the sediment affect L. variegatus more than MPs that are layered on the sediment surface. Kaolin as a natural, fine-sized particle control created somewhat favorable conditions for the worm. The main driver for toxicity, however, proved to be chemicals associated with the plastic product and its previous content.

Identification of Drug Resistance Determinants in a Clinical Isolate of Pseudomonas aeruginosa by High-Density Transposon Mutagenesis.

With the aim to identify potential new targets to restore antimicrobial susceptibility of multidrug-resistant (MDR) Pseudomonas aeruginosa isolates, we generated a high-density transposon (Tn) insertion mutant library in an MDR P. aeruginosa bloodstream isolate (isolate ID40). The depletion of Tn insertion mutants upon exposure to cefepime or meropenem was measured in order to determine the common resistome for these clinically important antipseudomonal ß-lactam antibiotics. The approach was validated by clean deletions of genes involved in peptidoglycan synthesis/recycling, such as the genes for the lytic transglycosylase MltG, the murein (Mur) endopeptidase MepM1, the MurNAc/GlcNAc kinase AmgK, and the uncharacterized protein YgfB, all of which were identified in our screen as playing a decisive role in survival after treatment with cefepime or meropenem. We found that the antibiotic resistance of P. aeruginosa can be overcome by targeting usually nonessential genes that turn essential in the presence of therapeutic concentrations of antibiotics. For all validated genes, we demonstrated that their deletion leads to the reduction of ampC expression, resulting in a significant decrease in ß-lactamase activity, and consequently, these mutants partly or completely lost resistance against cephalosporins, carbapenems, and acylaminopenicillins. In summary, the determined resistome may comprise promising targets for the development of drugs that may be used to restore sensitivity to existing antibiotics, specifically in MDR strains of P. aeruginosa.

Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system.

Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-ß-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.

Deprivation of the Periplasmic Chaperone SurA Reduces Virulence and Restores Antibiotic Susceptibility of Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the main causative agents of nosocomial infections and the spread of multidrug-resistant strains is rising. Therefore, novel strategies for therapy are urgently required. The outer membrane composition of Gram-negative pathogens and especially of Pa restricts the efficacy of antibiotic entry into the cell and determines virulence. For efficient outer membrane protein biogenesis, the ß-barrel assembly machinery (BAM) complex in the outer membrane and periplasmic chaperones like Skp and SurA are crucial. Previous studies indicated that the importance of individual proteins involved in outer membrane protein biogenesis may vary between different Gram-negative species. In addition, since multidrug-resistant Pa strains pose a serious global threat, the interference with both virulence and antibiotic resistance by disturbing outer membrane protein biogenesis might be a new strategy to cope with this challenge. Therefore, deletion mutants of the non-essential BAM complex components bamB and bamC, of the skp homolog hlpA as well as a conditional mutant of surA were investigated. The most profound effects for both traits were associated with reduced levels of SurA, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment and attenuation of virulence in a Galleria mellonella infection model. Strikingly, the depletion of SurA in a multidrug-resistant clinical bloodstream isolate re-sensitized the strain to antibiotic treatment. From our data we conclude that SurA of Pa serves as a promising target for developing a drug that shows antiinfective activity and re-sensitizes multidrug-resistant strains to antibiotics.

Cholesterol and host cell surface proteins contribute to cell-cell fusion induced by the Burkholderia type VI secretion system 5.

Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 (T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl- ß-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell.

Effectiveness of interventions for adults with mild to moderate intellectual disabilities and mental health problems: systematic review and meta-analysis.

BACKGROUND: There is a lack of available evidence in relation to the effectiveness of interventions for adults with mild to moderate intellectual disability and mental health problems. AIMS: To evaluate the efficacy of interventions for adults with mild to moderate intellectual disabilities and co-occurring mental health problems. METHOD: An electronic literature search of the databases Medline, EMBASE, PsycINFO and EBM Reviews aimed at identifying randomised controlled trials (RCTs) and controlled trials testing any type of intervention (psychotherapy, biological or system level) for people with mild to moderate intellectual disabilities (IQ score 35-69) targeting comorbid mental health problems. Additionally a meta-analysis was conducted. RESULTS: Twelve studies met the inclusion criteria. No significant effect was found for the predefined outcome domains behavioural problems, depression, anxiety, quality of life and functioning. The effect size for depression (d = 0.49) was moderate but non-significant. Quality of studies was moderate and heterogeneity was high. CONCLUSIONS: There is no compelling evidence supporting interventions aiming at improving mental health problems in people with mild to moderate intellectual disability. The number of available trials is too low for definite conclusions. Some interventions are promising and should be evaluated further in larger and more rigorous trials.

Increased inducible nitric oxide synthase and arginase II expression in heart failure: no net nitrite/nitrate production and protein S-nitrosylation.

Our objective was to address the balance of inducible nitric oxide (NO) synthase (iNOS) and arginase and their contribution to contractile dysfunction in heart failure (HF). Excessive NO formation is thought to contribute to contractile dysfunction; in macrophages, increased iNOS expression is associated with increased arginase expression, which competes with iNOS for arginine. With substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS). In rabbits, HF was induced by left ventricular (LV) pacing (400 beats/min) for 3 wk. iNOS mRNA [quantitative real-time PCR (qRT-PCR)] and protein expression (confocal microscopy) were detected, and arginase II expression was quantified with Western blot; serum arginine and myocardial nitrite and nitrate concentrations were determined by chemiluminescence, and protein S-nitrosylation with Western blot. Superoxide anions were quantified with dihydroethidine staining. HF rabbits had increased LV end-diastolic diameter [20.0 + or - 0.5 (SE) vs. 17.2 + or - 0.3 mm in sham] and decreased systolic fractional shortening (11.1 + or - 1.4 vs. 30.6 + or - 0.7% in sham; both P < 0.05). Myocardial iNOS mRNA and protein expression were increased, however, not associated with increased myocardial nitrite or nitrate concentrations or protein S-nitrosylation. The serum arginine concentration was decreased (124.3 + or - 5.6 vs. 155.4 + or - 12.0 micromol/l in sham; P < 0.05) at a time when cardiac arginase II expression was increased (0.06 + or - 0.01 vs. 0.02 + or - 0.01 arbitrary units in sham; P < 0.05). Inhibition of iNOS with 1400W attenuated superoxide anion formation and contractile dysfunction in failing hearts. Concomitant increases in iNOS and arginase expression result in unchanged NO species and protein S-nitrosylation; with substrate limitation, uncoupled iNOS produces superoxide anions and contributes to contractile dysfunction.