Glucose-6-phosphate isomerase (GPI, EC 5.3.1.9) is a dimeric enzyme that catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate, the second reaction step of glycolysis. GPI deficiency, transmitted as an autosomal recessive trait, is considered the second most common erythro-enzymopathy of anaerobic glycolysis, after pyruvate kinase deficiency. Despite this, this defect may sometimes be misdiagnosed and only about 60 cases of GPI deficiency have been reported. GPI deficient patients are affected by chronic non-spherocytic hemolytic anemia of variable severity; in rare cases, intellectual disability or neuromuscular symptoms have also been reported. The gene locus encoding GPI is located on chromosome 19q13.1 and contains 18 exons. So far, about 40 causative mutations have been identified. We report the clinical, hematological and molecular characteristics of 12 GPI deficient cases (eight males, four females) from 11 families, with a median age at admission of 13 years (ranging from 1 to 51); eight of them were of Italian origin. Patients displayed moderate to severe anemia, that improves with aging. Splenectomy does not always result in the amelioration of anemia but may be considered in transfusion-dependent patients to reduce transfusion intervals. None of the patients described here displayed neurological impairment attributable to the enzyme defect. We identified 13 different mutations in the GPI gene, six of them have never been described before; the new mutations affect highly conserved residues and were not detected in 1000 Genomes and HGMD databases and were considered pathogenic by several mutation algorithms. This is the largest series of GPI deficient patients so far reported in a single study. The study confirms the great heterogeneity of the molecular defect and provides new insights on clinical and molecular aspects of this disease.

The understanding that fluid ingestion attenuates thermoregulatory and circulatory stress during exercise in the heat was based on studies conducted in relatively dry (∼50% RH) environments. It remains undetermined whether similar effects occur during exercise in a warm and more humid environment, where evaporative capacity is reduced. Nine well-trained, unacclimatised male runners were randomly assigned to perform four experimental trials where they ran for 60 min at an intensity of 70% VO2max followed by an incremental exercise test until volitional exhaustion. The four trials consisted of non-fluid ingestion (NF) and fluid ingestion (FI) in a warm-dry (WD) and warm-humid condition (WH). Time to exhaustion (TTE), body temperature (Tb), whole body sweat rate, partitional calorimetry measures, heart rate and plasma volume were recorded during exercise. There was no significant difference in Tb following 60 min of exercise in FI and NF trial within both WD (37.3°C ± 0.4 vs. 37.4°C ± 0.3; p > 0.05) and WH conditions (38.0°C ± 0.4 vs. 38.1°C ± 0.4; p > 0.05). The TTE was similar between FI and NF trials in both WH and WD, whereas exercise capacity was significantly shorter in WH than WD (9.1 ± 2.8 min vs. 12.7 ± 2.4 min, respectively; p = 0.01). Fluid ingestion failed to provide any ergogenic benefit in attenuating thermoregulatory and circulatory stress during exercise in the WH and WD conditions. Consequently, exercise performance was not enhanced with fluid ingestion in the warm-humid condition, although the humid environment detrimentally affected exercise endurance.

Intrauterine growth restriction (IUGR) is a condition where the fetus does not achieve optimal growth, commonly caused by placental insufficiency. The chronic decrease in blood flow restricts oxygen and nutrient supply to the fetus, which can damage numerous organ systems, with the fetal brain being particularly vulnerable. Although white matter and neuronal injury are evident in IUGR infants, the specific mechanisms underlying these changes are poorly understood. Inflammation is considered to be a main driver in exacerbating brain injury. Using a spontaneous piglet model of IUGR, we aim to determine whether administration of the anti-inflammatory drug ibuprofen will decrease inflammation at postnatal day 4 (P4). The treatment group received ibuprofen (20 mg/kg/day on day 1 and 10 mg/kg/day on days 2 and 3) in piglet formula during the morning feed each day and brains examined on P4. Markers of inflammation, apoptosis, cell proliferation, neuronal injury, and white matter injury were examined. Ibuprofen treatment ameliorated the increase in numbers of microglia and astrocytes in the parietal cortex and white matter tracts of the IUGR piglet brain on P4 as well as decreasing proinflammatory cytokines. Ibuprofen treatment prevented the reduction in apoptosis, neuronal cell counts, and myelin index in the IUGR piglets. Our findings demonstrate ibuprofen reduces the inflammatory response in the IUGR neonatal brain and concurrently reduces neuronal and white matter impairment.

In the context of intensive aquaculture, meagre (Argyrosomus regius) is one of the most important new aquaculture species in Southern Europe and several studies are focused on the optimization of its culture. Nevertheless, stressors such as handling during transport or culture maintenance may affect the immune system, thereby impairing some immune responses or provoking cellular damage. One strategy that has been used to avert this type of negative stress response is the supplementation of amino acids to improve resistance to stress. In this experiment, meagre (105.0 ± 2.6 g, mean ± standard deviation) juveniles were fed two diets for a period of 7 days, the first a commercial diet supplemented with 1% tryptophan (Trp) and second, the same commercial diet without tryptophan supplementation (control group). The effects of two types of handling stressors (air exposure and confinement/netting) on fish fed both diets was evaluated in terms of gene expression of the selected gut immunity markers, such as (1) innate immune response processes: c3 complement (c3), lysozyme (lys), and cyclooxygenase (cox2); (2) humoral immune response processes: interferon type 1 (ifn1), mx protein (mxp), interleukin 1b (il-1b), tumor necrosis factor 1a (tnf1a), and interleukin 10 (il-10); (3) antimicrobial peptides: defensin (def), hepcidin (hep), piscidin (pis), and a marker for mitochondrial respiration: glyceraldehyde 3-phosphate dehydrogenase (gapdh). Samples of the anterior intestine were collected at 1 and 6 h post-stress (hps). Results showed that in fish fed 1% Trp, the air exposure resulted in an upregulation of gene expression at 6 hps for c3, lys, cox2, ifn1, mxp, il-10 and gapdh, and il-1b and pis. The confinement/netting test for fish fed 1% Trp resulted in an upregulation of c3 and mxp and a downregulation of cox2, ifn1, il-1b, tnf1a, il-10, def, hep, and gapdh at both post-stress times (1 and 6 hps). According to the present study, dietary supplementation with 1% Trp may be considered as a proper nutritional strategy for improving tolerance and/or alleviating acute response to handling stressors.

Objective: Gabapentin (GBP) is an anticonvulsant medication that is also used to treat restless legs syndrome (RLS) and posttherapeutic neuralgia. GBP is commonly prescribed off-label for psychiatric disorders despite the lack of strong evidence. However, there is growing evidence that GBP may be effective and clinically beneficial in both psychiatric disorders and substance use disorders. This review aimed to perform a systematic analysis of peer-reviewed published literature on the efficacy of GBP in the treatment of psychiatric disorders and substance use disorders. Methods: This review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The PubMed and Ovid MEDLINE literature databases were screened and filtered by using specific search terms and inclusion/exclusion criteria. The full texts of selected studies were subsequently retrieved and reviewed. The search terms generated 2,604 results from the databases. After excluding all duplicates, 1,088 citations were left. Thereafter, we applied inclusion and exclusion criteria; a total of 54 papers were retained for detailed review. Results: This literature review concludes that GBP appears to be effective in the treatment of various forms of anxiety disorders. It shows some effectiveness in bipolar disorder as an adjunctive therapeutic agent, while the evidence for monotherapy is inconclusive. In substance use disorders, GBP is effective for acute alcohol withdrawal syndrome (AWS) with mild to moderate severity; it reduces cravings, improves the rate of abstinence, and delays return to heavy drinking. GBP may have some therapeutic potential in the treatment of opioid addiction and cannabis dependence, but there is limited evidence to support its use. No significant benefit of GBP has been conclusively observed in the treatment of OCD, PTSD, depression, or cocaine and amphetamine abuse. Conclusion: GBP appears to be effective in some forms of anxiety disorders such as preoperative anxiety, anxiety in breast cancer survivors, and social phobia. GBP has shown to be safe and effective in the treatment of alcohol dependence. However, the literature suggests that GBP is effective as an adjunctive medication rather than a monotherapy. More clinical trials with larger patient populations are needed to support gabapentin's off-label use in psychiatric disorders and substance use disorders. It is worth noting that numerous clinical studies that are discussed in this review are open-label trials, which are inherently less rigorously analyzed. Therefore, more extensive investigations are required to examine not only the efficacy of GBP, but also its safety and tolerance.

Central precocious puberty (CPP) has been shown to exert significant effects on psychosocial development. These early puberty-related hormones and psychosocial functional changes are considered to be associated with specific brain development. However, the biological mechanisms underlying the sculpting of human brain architecture and modulation of psychosocial transformation by puberty-related hormonal maturation remain elusive, especially during the early phase of CPP. The current investigation aims to specify the brain regions in which early hormone-related maturation effects occur during CPP and their relationships with psychological functions. 65 young girls (aged 4.3-8.0 years) underwent structural imaging on a 3T MR system, completed psychological tests and performed the gonadotropin-releasing hormone (GnRH) stimulation test to identify hormonal manifestations of hypothalamic-pituitary-gonadal axis (HPG axis) activation. Based on the GnRH test, 28 young girls were identified with CPP, whereas the other 37 girls were identified with non-central precocious puberty (NCPP). Cortical parameters were calculated and compared between the two groups after adjusting for age, weight, and height. Brain regions showing group differences were extracted and correlated with serum hormone levels and psychological parameters. The CPP girls showed thinner cortices primarily in the right rostral middle frontal cortex. This morphological difference was positively correlated with stimulated estradiol (E2) levels. Further, higher E2 levels were significantly associated with higher hyperactivity scores. Premature HPG axis activation in CPP girls at an early stage appears to exert remodeling effects on brain anatomy, primarily in the prefrontal cortex, which may affect psychological development following the emergence of robust changes in sex hormones.

This research presents the development of a short scale named "NOTECHS+" to measure the Non-Technical Skills (i.e., NOTECHS: Cooperation, Leadership and Managerial skills, Decision-Making, and Situational Awareness), Resilience and Emotion Regulation, in a sector that comprises the aviation and the emergency personnel: the Helicopter Emergency Medical Service (HEMS). The design process of the scale was carried out starting from a review on the behavioral markers used to detect the NOTECHS. Moreover, 70 interviews with HEMS experts have been conducted with the aim of developing Resilience and Emotion Regulation items by considering the different professional profiles (e.g., pilots, nurses, physicians, etc.) which compose the HEMS. Through a pre-assessment procedure, a Q-Sort test was performed on a sample of students (n = 30) to test the logical principles, but also intelligibility and clarity, of the items developed. Once the instrument was defined, 211 participants from the HEMS sector were surveyed to test the theoretical model behind the NOTECHS+ instrument. First exploratory and then confirmatory analysis yielded results that suggested that the 18 items selected conform to a bi-factor model composed of three skill-dimensions: Social skills (i.e., Cooperation, and Leadership and Managerial skills), Cognitive skills (i.e., Decision-Making and Situational Awareness) and Emotional skills (i.e., Resilience and Emotional Regulation). Finally, the study ends with a discussion on the results obtained, including practical implications on assessment and training based on this novel instrument.

The present qualitative study analyzes how a group of young people already involved in STEM fields perceive the prototypical person working in STEM. Gender differences between participants in technological and non-technological STEM fields were analyzed. A total of 27 young people (59.3% women) took part in the interviews (Mean Age = 25.48 years). Of them, 16 participants were working in STEM professions, and 11 were enrolled in the final courses of STEM degrees. The results of the content analysis were examined in light of social role theory and the multidimensional structure of gender stereotypes. Men in these fields were therefore attributed an unappealing and weird physical appearance. Some female participants linked STEM professionals' intellectual abilities to the stereotype that men have higher abilities in these fields. Whereas females attributed effort and perseverance to STEM professionals' intellectual aptitudes, males referred to the development of soft skills. Participants in technological STEM fields connected the stereotype of being a 'weirdo' to a boring job, whereas those in non-technological fields linked it to their unconventional character. Some participants were disappointed by a lack of correspondence between expectations and the actual job STEM professionals do. Moreover, females in technological STEM fields commented on the job's low social impact, while males mentioned low attainment of technical qualifications. Most referents in STEM fields were masculine, some of whom were present in the mass media. The practical implications of the findings are discussed.

Human movement based on sensory control is significant to motor task performance. Thus, impairments to sensory input significantly limit feedback-type motor control. The present study introduces a vibrotactile biofeedback (BF) system which augments information regarding the user's foot pressure to enhance gait performance. The effects of the proposed system on the gait patterns of healthy older adults and on the cognitive load during gait were evaluated; these factors are essential to clarify feasibility of the device in real-life settings. The primary task of our study was to evaluate gait along with a cognitively demanding activity in 10 healthy older adults. Regarding kinematic and kinetic data in the BF condition, the subjects had significantly increased ankle dorsiflexion during the heel contact phase in the sagittal plane and marginally increased foot pressure at the toe-off and stride length. However, such kinematic and kinetic changes were not attributed to the increased walking speed. In addition, cognitive performance (i.e., the number of correct answers) was significantly decreased in participants during gait measurements in the BF condition. These data suggest that the system had the potential for modifying the kinematic and kinetic patterns during walking but not the more comprehensive walking performance in older adults. Moreover, the device appears to place a cognitive load on older adults. This short report provides crucial primary data that would help in designing successful sensory augmentation devices and further research on a BF system.

Objective: From a dynamic system approach, this study evaluated the impact of a new training protocol using a mechanical horse on the postural coordination of brain-damaged patients. Methods: Eighteen volunteer brain-damaged patients (i.e., post-stroke or traumatic brain injury) were recruited and randomly divided into an experimental group (horse group; n = 10, conventional therapy associated with horse-riding exercise on the mechanical horse for 30 min, twice a week, for 12 weeks) and a control group (n = 8; conventional therapy without intervention on the mechanical horse). Postural coordination was evaluated during pre- and post-tests through discrete relative phase (DRP) computation: ϕHead-Horse, ϕTrunk-Horse. Results: A significant effect of used training has been showed, F (1, 15) = 16.6 (p < 0.05) for all patients, concerning the trunk/horse coordination. Conclusion: This pilot study results showed the impact of this new training method on the postural coordination of these patients. After 24 sessions, the coordination of the horse group patients differed from that of the control group, showing their ability to adapt to constraints and develop specific modes of postural coordination (trunk/horse antiphase) to optimize their posture.

Objective: Determine if NC001, an oral formulation of nicotine that reduces levodopa-induced dyskinesias (LIDs) in MPTP-Parkinson monkeys, could reduce falls, freezing of gait (FOG), and LIDs in Parkinson disease (PD) patients. Methods: Previously collected data from a study analyzing the effects of NC001 on LIDs in PD patients were reanalyzed. Because indirect-acting cholinergic drugs are sometimes helpful in reducing falls, we hypothesized that NC001, a direct-acting cholinergic agonist, could reduce falls in PD. The original 12-center, double-blind, randomized trial enrolled 65 PD patients. NC001 or placebo was administered 4 times per day for 10 weeks, beginning at 4 mg/day and escalating to 24 mg/day. Assessments included the Unified Dyskinesia Rating Scale (UDysRS) and Parts II-III of the original Unified Parkinson's Disease Rating Scale (UPDRS). Results: Randomization (1:1) resulted in 35 patients on NC001 and 30 on placebo at baseline. Thirty and 27 patients, respectively, had data available for an intent-to-treat analysis. NC001 was safe and well-tolerated. After 10 weeks, NC001 patients (14/30) had a significant reduction in falls vs. placebo patients (3/27) (p = 0.0041) as assessed by UPDRS Part II. NC001 patients (12/30) also had significantly reduced FOG vs. placebo patients (4/27) (p = 0.0043). NC001 patients, compared with placebo patients, had a significant improvement (p = 0.01) in UDysRS ambulation subtest (40% vs. 3%, respectively). Although NC001 patients had a greater reduction in dyskinesias on the UDysRS than placebo patients (30% vs. 19%, respectively), this was not significant (p = 0.09). Conclusions: NC001 significantly improved two refractory symptoms of PD, falls and FOG. The reduction in falls and FOG is attributed to selective stimulation of nicotinic receptors. Clinical Trial Registration: Conducted under IND 105, 268, serial number 0000. ClinicalTrials.gov identifier NCT00957918.

Laminin-211 deficiency leads to the most common form of congenital muscular dystrophy in childhood, MDC1A. The clinical picture is characterized by severe muscle weakness, brain abnormalities and delayed motor milestones defining MDC1A as one of the most severe forms of congenital muscular diseases. Although the molecular genetic basis of this neurological disease is well-known and molecular studies of mouse muscle and human cultured muscle cells allowed first insights into the underlying pathophysiology, the definition of marker proteins in human vulnerable tissue such as skeletal muscle is still lacking. To systematically address this need, we analyzed the proteomic signature of laminin-211-deficient vastus muscle derived from four patients and identified 86 proteins (35 were increased and 51 decreased) as skeletal muscle markers and verified paradigmatic findings in a total of two further MDC1A muscle biopsies. Functions of proteins suggests fibrosis but also hints at altered synaptic transmission and accords with central nervous system alterations as part of the clinical spectrum of MDC1A. In addition, a profound mitochondrial vulnerability of the laminin-211-deficient muscle is indicated and also altered abundances of other proteins support the concept that metabolic alterations could be novel mechanisms that underline MDC1A and might constitute therapeutic targets. Intersection of our data with the proteomic signature of murine laminin-211-deficient gastrocnemius and diaphragm allowed the definition of nine common vulnerable proteins representing potential tissue markers.

Sport-related concussion is an increasingly common injury among adolescents, with repetitive mild traumatic brain injuries (RmTBI) being a significant risk factor for long-term neurobiological and psychological consequences. It is not uncommon for younger professional athletes to consume anabolic-androgenic steroids (AAS) in an attempt to enhance their performance, subjecting their hormonally sensitive brains to potential impairment during neurodevelopment. Furthermore, RmTBI produces acute neuroendocrine dysfunction, specifically in the anterior pituitary, disrupting the hypothalamic-pituitary adrenal axis, lowering cortisol secretion that is needed to appropriately respond to injury. Some AAS users exhibit worse symptoms post-RmTBI if they quit their steroid regime. We sought to examine the pathophysiological outcomes associated with the abrupt cessation of the commonly abused AAS, Metandienone (Met) on RmTBI outcomes in rats. Prior to injury, adolescent male rats received either Met or placebo, and exercise. Rats were then administered RmTBIs or sham injuries, followed by steroid and exercise cessation (SEC) or continued treatment. A behavioral battery was conducted to measure outcomes consistent with clinical representations of post-concussion syndrome and chronic AAS exposure, followed by analysis of serum hormone levels, and qRT-PCR for mRNA expression and telomere length. RmTBI increased loss of consciousness and anxiety-like behavior, while also impairing balance and short-term working memory. SEC induced hyperactivity while Met treatment alone increased depressive-like behavior. There were cumulative effects whereby RmTBI and SEC exacerbated anxiety and short-term memory outcomes. mRNA expression in the prefrontal cortex, amygdala, hippocampus, and pituitary were modified in response to Met and SEC. Analysis of telomere length revealed the negative impact of SEC while Met and SEC produced changes in serum levels of testosterone and corticosterone. We identified robust changes in mRNA to serotonergic circuitry, neuroinflammation, and an enhanced stress response. Interestingly, Met treatment promoted glucocorticoid secretion after injury, suggesting that maintained AAS may be more beneficial than abstaining after mTBI.

Background: We compared resting-state functional connectivity (RSFC) among limbic and temporal lobe regions between patients with medial temporal lobe epilepsy (mTLE) and healthy control subjects to identify imaging evidence of functional networks related to seizure frequency, age of seizure onset, and duration of epilepsy. Methods: Twelve patients with drug-resistant, unilateral medial temporal lobe epilepsy and 12 healthy control subjects matched for age, sex, and handedness participated in the imaging experiments. We used network-based statistics to compare functional connectivity graphs in patients with mTLE and healthy controls to investigate the relationship between functional connectivity abnormalities and seizure frequency. Results: Among mTLE patients, we found functional network abnormalities throughout the limbic system, but primarily in the hemisphere ipsilateral to the seizure focus. The RSFCs between ipsilateral hypothalamus and ventral anterior cingulate cortex and between ipsilateral subiculum and contralateral posterior cingulate cortex were highly correlated with seizure frequency. Discussion: These findings suggest that in mTLE, changes in limbic networks ipsilateral to the epileptic focus are common. The pathological changes in connectivity between cingulate cortex, hypothalamus and subiculum ipsilateral to the seizure focus were correlated with increased seizure frequency.

Aspergillus fumigatus, a saprophytic filamentous fungus, is a serious opportunistic pathogen of mammals and it is the primary causal agent of invasive aspergillosis (IA). Mitogen activated protein Kinases (MAPKs) are important components involved in diverse cellular processes in eukaryotes. A. fumigatus MpkC and SakA, the homologs of the Saccharomyces cerevisiae Hog1 are important to adaptations to oxidative and osmotic stresses, heat shock, cell wall damage, macrophage recognition, and full virulence. We performed protein pull-down experiments aiming to identify interaction partners of SakA and MpkC by mass spectrometry analysis. In presence of osmotic stress with sorbitol, 118, and 213 proteins were detected as possible protein interactors of SakA and MpkC, respectively. Under cell wall stress caused by congo red, 420 and 299 proteins were detected interacting with SakA and MpkC, respectively. Interestingly, a group of 78 and 256 proteins were common to both interactome analysis. Co-immunoprecipitation (Co-IP) experiments showed that SakA::GFP is physically associated with MpkC:3xHA upon osmotic and cell wall stresses. We also validated the association between SakA:GFP and the cell wall integrity MAPK MpkA:3xHA and the phosphatase PtcB:3xHA, under cell wall stress. We further characterized A. fumigatus PakA, the homolog of the S. cerevisiae sexual developmental serine/threonine kinase Ste20, as a component of the SakA/MpkC MAPK pathway. The ΔpakA strain is more sensitive to cell wall damaging agents as congo red, calcofluor white, and caspofungin. Together, our data supporting the hypothesis that SakA and MpkC are part of an osmotic and general signal pathways involved in regulation of the response to the cell wall damage, oxidative stress, drug resistance, and establishment of infection. This manuscript describes an important biological resource to understand SakA and MpkC protein interactions. Further investigation of the biological roles played by these protein interactors will provide more opportunities to understand and combat IA.

The obligate intracellular pathogen Chlamydia trachomatis (Ctr) is the causative agent of the most common form of sexually transmitted disease in the United States. Genital infections with C. trachomatis can lead to inflammatory tissue damage followed by scarring and tissue remodeling during wound healing. Extensive scarring can lead to ectopic pregnancy or infertility. Classically activated macrophages (CA mϕ), with their anti-microbial effector mechanisms, are known to be involved in acute inflammatory processes during the course of infection. In contrast, alternatively activated macrophages (AA mϕ) contribute to tissue repair at sites of wound healing, and have reduced bactericidal functions. They are present during infection, and thus potentially can provide a growth niche for C. trachomatis during a course of infection. To address this question, macrophages derived from CD14-positive monocytes magnetically isolated from peripheral blood mononuclear cells (PBMC) were treated with interferon-γ or interleukin-4 to produce CA mϕ or AA mϕ, respectively. Confocal microscopy of chlamydial inclusions and quantification of infectious yields revealed better pathogen growth and development in AA mϕ than CA mϕ, which correlated with the reduced expression of indoleamine 2,3-dioxygenase, a known anti-chlamydial effector of the host. Furthermore, AA mϕ stained strongly for transferrin receptor and secreted higher amounts of anti-inflammatory interleukin-10 compared to CA mϕ, characteristics that indicate its suitability as host to C. trachomatis. CA, AA, and resting mϕ were infected with Ctr serovar L2. The data suggest that IL-10 produced by infected AA mϕ attenuated the anti-chlamydial function of CA mϕ with growth recovery observed in infected CA mϕ in the presence of infected, but not mock-infected AA mϕ. This could be related to our observation that IL-10 treatment of infected CA mϕ promoted better chlamydial growth. Thus, in addition to serving as an additional niche, AA mϕ might also serve as a means to modulate the immediate environment by attenuating the anti-chlamydial functions of nearby CA mϕ in a manner that could involve IL-10 produced by infected AA mϕ.

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter P dsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the P dsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the P dsrLL -mrfp fusion or the P dsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from P dsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from P dsrLL . A different behavior was detected, when expression from P dsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.

The bacterial endosymbiont Wolbachia interacts with different invertebrate hosts, engaging in diverse symbiotic relationships. Wolbachia is often a reproductive parasite in arthropods, but an obligate mutualist in filarial nematodes. Wolbachia was recently discovered in plant-parasitic nematodes, and, is thus far known in just two genera Pratylenchus and Radopholus, yet the symbiont's function remains unknown. The occurrence of Wolbachia in these economically important plant pests offers an unexplored biocontrol strategy. However, development of Wolbachia-based biocontrol requires an improved understanding of symbiont-host functional interactions and the symbiont's prevalence among nematode field populations. This study used a molecular-genetic approach to assess the prevalence of a Wolbachia lineage (wPpe) in 32 field populations of Pratylenchus penetrans. Populations were examined from eight different plant species in Washington, Oregon, and California. Nematodes were also screened for the endosymbiotic bacterium Cardinium (cPpe) that was recently shown to co-infect P. penetrans. Results identified wPpe in 9/32 and cPpe in 1/32 of P. penetrans field populations analyzed. No co-infection was observed in field populations. Wolbachia was detected in nematodes from 4/8 plant-hosts examined (raspberry, strawberry, clover, and lily), and in all three states surveyed. Cardinium was detected in nematodes from mint in Washington. In the wPpe-infected P. penetrans populations collected from raspberry, the prevalence of wPpe infection ranged from 11 to 58%. This pattern is unlike that in filarial nematodes where Wolbachia is an obligate mutualist and occurs in 100% of the host. Further analysis of wPpe-infected populations revealed female-skewed sex ratios (up to 96%), with the degree of skew positively correlating with wPpe prevalence. Uninfected nematode populations had approximately equal numbers of males and females. Comparisons of 54 wPpe 16S ribosomal RNA sequences revealed high similarity across the geographic isolates, with 45 of 54 isolates being identical at this locus. The complete absence of wPpe among some populations and low prevalence in others suggest that this endosymbiont is not an obligate mutualist of P. penetrans. The observed sex ratio bias in wPpe-infected nematode populations is similar to that observed in arthropods where Wolbachia acts as a reproductive manipulator, raising the question of a similar role in plant-parasitic nematodes.

Two-component systems (TCSs) are dominant regulating components in bacteria for responding to environmental stimuli. However, little information is available on how TCSs in Enterococcus faecium respond to bile salts - an important environmental stimulus for intestinal bacteria. In this study, the gene expression of 2 TCSs, BsrXRS and LiaFSR, was positively correlated with survival rates of different E. faecium isolates during exposure to ox gall. Moreover, gene disruptions of bsrR, bsrS, liaS, and liaR significantly reduced the survival rates of E. faecium in the presence of ox gall. Finally, EMSA results indicated that BsrR functioned as a transcription regulator for expression of its own gene as well as lipoate-protein ligase A (lplA). Additional 27 potential target genes by BsrR were revealed through in silico analyses. These findings suggest that BsrXRS and LiaFSR systems play important roles in bile salt resistance in E. faecium.

Plant pollen are an important source of antigens that evoke allergic responses. Protein antigens have been the focus of studies aiming to elucidate the mechanisms responsible for allergic reactions to pollen. However, proteins are not the sole active agent present in pollen. It is known that pollen grains contain lipids essential for its reproduction and bioactive lipid mediators. These small molecular compounds are co-delivered with the allergens and hence have the potential to modulate the immune response of subjects by activating their innate immune cells. Previous reports showed that pollen associated lipid mediators exhibited neutrophil- and eosinophil-chemotactic activity and induced polarization of dendritic cells (DCs) toward a Th2-inducing phenotype. In our study we performed chemical analyses of the pollen associated lipids, that are rapidly released upon hydration. As main components we have identified different types of phytoprostanes (PhytoPs), and for the first time phytofurans (PhytoFs), with predominating 16-F1t-PhytoPs (PPF1-I), 9-F1t-PhytoPs (PPF1-II), 16-E1t-PhytoPs (PPE1-I) and 9-D1t-PhytoPs (PPE1-II), and 16(RS)-9-epi-ST-Δ14-10-PhytoFs. Interestingly 16-E1t-PhytoP and 9-D1t-PhytoPs were found to be bound to glycerol. Lipid-containing samples (aqueous pollen extract, APE) induced murine mast cell chemotaxis and IL-6 release, and enhanced their IgE-dependent degranulation, demonstrating a role for these lipids in the immediate effector phase of allergic inflammation. Noteworthy, mast cell degranulation seems to be dependent on glycerol-bound, but not free phytoprostanes. On murine dendritic cells, APE selectively induced the upregulation of CD1d, likely preparing lipid-antigen presentation to iNKT cells. Our report contributes to the understanding of the activity of lipid mediators in the immediate effector phase of allergic reactions but identifies a yet undescribed pathway for the recognition of pollen-derived glycolipids by iNKT cells.