Molecular and neurochemical substrates of the audiogenic seizure strains: The GASH:Sal model.

PURPOSE: Animal models of audiogenic epilepsy are useful tools to understand the mechanisms underlying human reflex epilepsies. There is accumulating evidence regarding behavioral, anatomical, electrophysiological, and genetic substrates of audiogenic seizure strains, but there are still aspects concerning their neurochemical basis that remain to be elucidated. Previous studies have shown the involved of γ-amino butyric acid (GABA) in audiogenic seizures. The aim of our research was to clarify the role of the GABAergic system in the generation of epileptic seizures in the genetic audiogenic seizure-prone hamster (GASH:Sal) strain. MATERIAL AND METHODS: We studied the K+/Cl- cotransporter KCC2 and ß2-GABAA-type receptor (GABAAR) and ß3-GABAAR subunit expressions in the GASH:Sal both at rest and after repeated sound-induced seizures in different brain regions using the Western blot technique. We also sequenced the coding region for the KCC2 gene both in wild- type and GASH:Sal hamsters. RESULTS: Lower expression of KCC2 protein was found in GASH:Sal when compared with controls at rest in several brain areas: hippocampus, cortex, cerebellum, hypothalamus, pons-medulla, and mesencephalon. Repeated induction of seizures caused a decrease in KCC2 protein content in the inferior colliculus and hippocampus and an increase in the pons-medulla. When compared to controls, the basal ß2-GABAAR subunit in the GASH:Sal was overexpressed in the inferior colliculus, rest of the mesencephalon, and cerebellum, whereas basal ß3 subunit levels were lower in the inferior colliculus and rest of the mesencephalon. Repeated seizures increased ß2 both in the inferior colliculus and in the hypothalamus and ß3 in the hypothalamus. No differences in the KCC2 gene-coding region were found between GASH:Sal and wild-type hamsters. CONCLUSIONS: These data indicate that GABAergic system functioning is impaired in the GASH:Sal strain, and repeated seizures seem to aggravate this dysfunction. These results have potential clinical relevance and support the validity of employing the GASH:Sal strain as a model to study the neurochemistry of genetic reflex epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Lipid phase separation impairs membrane thickness sensing by the Bacillus subtilis sensor kinase DesK.

Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions. IMPORTANCE: The B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.

Quorum-sensing and BvrR/BvrS regulation, the type IV secretion system, cyclic glucans, and BacA in the virulence of Brucella ovis: similarities to and differences from smooth brucellae.

Brucella ovis is a rough bacterium--lacking O-polysaccharide chains in the lipopolysaccharide--that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguish B. ovis from smooth Brucella, which require O-polysaccharide chains for virulence, we have analyzed the significance in B. ovis of the main virulence factors described for smooth Brucella. Attempts to obtain strains of virulent B. ovis strain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system for in vitro survival, while the inactivation of bacA--in contrast to the results seen with smooth Brucella--did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake of B. ovis PA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by the virB operon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described for B. melitensis, VjbR regulates the transcription of the virB operon positively, and the N-dodecanoyl-dl-homoserine lactone (C(12)-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of the fliF flagellar gene was observed in B. ovis, probably due to the two deletions detected upstream of fliF. These results, together with others reported in the text, point to similarities between rough virulent B. ovis and smooth Brucella species as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics of B. ovis.

Resuscitation with albumin using BET formula keeps at bay fluid administration in burned patients. An observational study.

BET (Biological Engineering Technology) formula uses fluids with high albumin concentration to resuscitate burn patients. It estimates fluid resuscitation as a function of Body Burned Surface Area (BBSA) (ml/h = BBSA (m2) × 220) and administers it through a combination of lactated ringer and 20% Albumin starting at a 1:1 relationship. The proportion of albumin is decreased every 8 h, and infusion rate is modified according to urinary output. The study's purpose was to review resuscitation related variables of all burned patients treated in our unit using BET formula. We retrospectively analyzed all patients admitted to our critical care burn unit during a five year period. Only those admitted within the first 12 h post-burn injury were considered. 40 patients met all inclusion criteria. Resuscitation volume during the first 24 h was 2.58 ml/kg/%BBSA, significantly less than Parkland's estimation (4 ml/kg/%BBSA; P < 0.05). Patients were successfully resuscitated showing a significant base excess increase and lactate clearance during the resuscitation period (base excess 120%; lactate 29%; P < 0.05). Burn related complications where: ARDS 27%, renal dysfunction 53%, wound deepening 20%, abdominal compartment syndrome 4.5%. In conclusion, BET formula is capable of resuscitating burn patients successfully, limiting fluid administration.

Heterozygous Genotype rs17580 AT (PiS) in SERPINA1 is Associated with COPD Secondary to Biomass-Burning and Tobacco Smoking: A Case-Control and Populational Study.

Purpose: The protease inhibitor S (PiS) and Z (PiZ) variants have been stated as the only genetic cause of chronic obstructive pulmonary disease (COPD) in Caucasians. However, its frequency in admixed populations is low. We aimed to identify genetic susceptibility between PiS (rs17580) and PiZ (rs28929474) polymorphisms with COPD related to tobacco smoking and biomass-burning smoke as well as to determine its frequencies in Mestizo and Amerindian populations from Mexico. Patients and Methods: One thousand and eight hundred seventy-eight subjects were included in two comparisons of cases and controls, (1) smokers with and without COPD (COPD-S, n=399; SWOC, n=1106); (2) Biomass-burning smoke-exposed subjects with and without COPD (COPD-BS, n=98; BBES, n=275). In addition, 2354 Mexican subjects identified as Mestizos (n=1952) and Amerindian (n=402) were included. The population structure was evaluated using 59 informative ancestry markers. Results: The AT genotype of rs17580 is associated with COPD in both comparisons (COPD-S vs SWOC p<0.001, OR=2.16; COPD-BS vs BBES p<0.0001, OR=11.50). The population of the Mexico-North has a greater Caucasian contribution (54.7%) compared to the center (46.9%) and southeast (42.7%). Conclusion: The rs17580, AT genotype, is associated with COPD in Mexican-Mestizo smokers and exposed to biomass-burning smoke. The rs17580 AT is more frequent in the Mexican-Mestizo population of the North of the country, which has a high Caucasian component.

Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species.

Members of the Omp25/Omp31 family of surface proteins were previously shown to participate in the virulence of some Brucella species and a different distribution of the seven proteins of this family among species could be related to the difference in pathogenicity and host preference they exhibit. Accordingly, in this work we have analyzed the expression of the genes coding for the Omp25/Omp31 family in the six classical Brucella species and a set of B. ovis mutant strains with each omp gene inactivated. Immunoblot of whole-cell lysates with antibodies raised against the purified recombinant outer membrane proteins (OMPs) did not show the simultaneous presence of the seven OMPs in any of the Brucella strains studied, but different Omp25/Omp31 profiles were detected, in our experimental conditions, between the Brucella strains representative of the six classical species. Transcripts for omp31, omp25 and omp25c were, in general, the most abundant of the family and some hits were found in B. ovis for a posttranscriptional regulation mechanism and for a compensatory mechanism increasing the synthesis of a protein to compensate for the absence of another one. Finally, the potential interest of Omp25c and Omp31b as subcellular vaccines, considering their occurrence in the Brucella strains studied and their antigenic relatedness with other proteins of the family, is discussed.

Importance of the Omp25/Omp31 family in the internalization and intracellular replication of virulent B. ovis in murine macrophages and HeLa cells.

The role of the outer membrane proteins of the Omp25/Omp31 family in invasiveness and intracellular survival of virulent B. ovis in phagocytes was analyzed. The absence of Omp25d or Omp22 in B. ovis abolished its invasive capacity in HeLa cells and reduced it in J774.A1 cells. Additionally, in J774.A1 cells, the Deltaomp25d mutant was unable to multiply, whereas the Deltaomp22 mutant was cleared at 24h post-infection. These findings demonstrate that Omp25d and Omp22 are essential for the invasion and survival of B. ovis inside host cells, and justify the strong attenuation in virulence of the Deltaomp25d and Deltaomp22 mutants.

Injection Laryngoplasty Using Autologous Fat Enriched with Adipose-Derived Regenerative Stem Cells: A Safe Therapeutic Option for the Functional Reconstruction of the Glottal Gap after Unilateral Vocal Fold Paralysis.

BACKGROUND: Paralysis of one vocal fold leads to glottal gap and vocal fold insufficiency that has significant impact upon a patient's quality of life. Fillers have been tested to perform intracordal injections, but they do not provide perdurable results. Early data suggest that enriching fat grafts with adipose-derived regenerative cells (ADRCs) promote angiogenesis and modulate the immune response, improving graft survival. The aim of this study is to propose ADRC-enriched adipose tissue grafts as effective filler for the paralyzed vocal fold to use it for functional reconstruction of the glottal gap. METHOD: This is the first phase I-IIA clinical trial (phase I/IIA clinical trial, unicentric, randomized, controlled, and two parallel groups), to evaluate the safety of a new therapy with ADRC-enriched fat grafting (ADRC: group I) for laryngoplasty after unilateral vocal fold paralysis. Control group patients received centrifuged autologous fat (CAF: group II) grafts. Overall mean age is 52.49 ± 16.60 years. Group I (ADRC): 7 patients (3 males and 4 females), 52.28 ± 20.95 year. Group II (CAF): 7 patients (3 males and 4 females), 52.71 ± 12.59 year. RESULTS: VHI-10 test showed that preoperative mean score was 24.21 ± 8.28. Postoperative mean score was 6.71 ± 6.75. Preoperative result in group I was 21.14 ± 3.58 and postoperative result was 3.14 ± 3.53. Preoperative result for group II was 27.29 ± 10.66. Postoperative score in group II was 10.29 ± 7.52. Wilcoxon and the Student t-tests showed that the patient's self-perception of posttreatment improvement is larger when ADRCs are used. Comparing pre- and posttreatment voice quality analysis, group I showed a p = 0.053. Group II showed a p = 0.007. There would be no significant differentiation between pre- and posttreatment results. This is true for group II and limited for group I. CONCLUSIONS: This prospective trial demonstrates the safety and efficacy of the treatment of glottal gap defects utilizing ADRC-enriched fat grafts. This trial is registered with NCT02904824.

Bioactive sol-gel glasses with and without a hydroxycarbonate apatite layer as substrates for osteoblast cell adhesion and proliferation.

The biocompatibility of three sol-gel bioactive glasses with SiO(2) as the main constituent (75, 72.5 and 70 mol%), identical CaO content (25mol%), and without or with P(2)O(5) as third constituent (0, 2.5 and 5 mol%), have been analyzed (S75, S72.5P2.5, and S70P5 glasses). These studies were performed on both untreated glasses and on glasses coated with a hydroxycarbonate apatite (HCA) layer formed in vitro by soaking 7d in an acellular simulated body fluid. Cell attachment, spreading and proliferation were studied using neonatal rat calvaria osteoblasts. Cells attach to the three untreated glasses but show a higher efficiency on that with the higher phosphate content (S70P5). The formation of the HCA layer significantly enhances this process (1.7-fold). In all cases, attachment is followed by cell spreading on the surface of the materials, adopting the cells a flattened morphology and showing diverse anchoring cell projections. Mitotic activity has been detected on osteoblasts growing on the sol-gel glasses, being this process 2-4-fold higher when the apatite-like layer is already formed. Taking into account the results herein presented, these bioactive glasses can be considered biocompatible. In addition, their biocompatibility is greatly enhanced after induction of the formation of an HCA layer.

Opposite caudal versus rostral brain nitric oxide synthase response to generalized seizures in a novel rodent model of reflex epilepsy.

AIMS: Nitric oxide (NO) is synthesized from L-arginine (L-Arg) by three different isoforms of NO synthase (NOS), i.e. the constitutive neuronal and endothelial NOS (nNOS and eNOS) and the inducible NOS (iNOS). NO has been involved in the pathophysiology of epilepsy, but available data are conflicting and the actual role of NO in epilepsy still remains to be clarified. In this study we investigated the basal and post-seizure levels of constitutive NOS (cNOS) activity as well as the expression of the cNOS isoforms across brain regions in a novel model of epilepsy. MAIN METHODS: cNOS activity was assessed in various brain areas along the rostro-caudal axis in control wild type hamsters, unstimulated generalized audiogenic seizure prone hamsters, Salamanca strain, GASH:Sal and GASH:Sal after 10 sound-induced epileptic seizures. Additionally, Western blot experiments for nNOS and eNOS were performed in those areas where relevant changes in cNOS activity were found. KEY FINDINGS: In the GASH:Sal, cNOS activity increased in the mesencephalic areas studied while cNOS activity decreased in both the striatum and cerebral cortex after 10 sound-induced epileptic seizures. nNOS (but not eNOS) expression paralleled the variations in cNOS activity. The same sound stimulation had no effect on control hamsters. SIGNIFICANCE: These results suggest a different NOS response in the regions close to the original epileptic focus (caudal, in our auditory model) versus the remote areas (rostral) possibly recruited at later stages or after repeated crises. These findings may account for some of the discrepancies found regarding the role of NO in epilepsy.

Differences in the outer membrane-related properties of the six classical Brucella species.

Outer membrane-related properties (such as auto-agglutination and susceptibility to various compounds) of strains representative of the six classical species of the genus Brucella were assessed. The differences identified could not be fully explained based on the smooth or rough phenotype of the strain. Smooth strains of the closely related species Brucella melitensis and B. abortus exhibited different susceptibility patterns and the rough, virulent B. ovis and B. canis strains were equally or more resistant to conditions such as pH, non-immune serum, hydrogen peroxide and bactericidal cationic peptides than smooth strains. Such heterogeneity in outer membrane characteristics could account for differences in pathogenicity and host tropism.

Cholesterol, ganglioside GM1 and class A scavenger receptor contribute to infection by Brucella ovis and Brucella canis in murine macrophages.

The establishment of infection by Brucella ovis and Brucella canis in J774.A1 macrophages was found to be dependent upon cholesterol and ganglioside GM(1), two components of lipid rafts. This process also required a class A scavenger receptor of macrophages, and was not inhibited by smooth and rough lipopolysaccharides from Brucella spp. In response to infection, both bacteria induced a weak degree of macrophage activation. These results demonstrate that B. ovis and B. canis use cell surface receptors common to smooth Brucella spp. for macrophage infection, thus limiting macrophage activation and favouring intracellular multiplication and/or the survival of both bacteria.

Experimental arthritis inhibits the insulin-like growth factor-I axis and induces muscle wasting through cyclooxygenase-2 activation.

Chronic arthritis induces cachexia associated with an inhibition of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system and an activation of the E3 ubiquitin-ligating enzymes muscle atrophy F-box (MAFbx) and muscle Ring finger 1 (MuRF1) in the skeletal muscle. The aim of this work was to study the role of cyclooxygenase (COX)-2 in chronic arthritis-induced cachexia. Arthritis was induced in rats by Freund's adjuvant injection, and the effects of two COX inhibitors (indomethacin, a nonspecific inhibitor, and meloxicam, a selective COX-2 inhibitor on pituitary GH and on liver and serum IGF-I levels) were tested. Arthritis decreased body weight gain and GH and liver IGF-I gene expression. In the arthritic rats, both inhibitors, indomethacin and meloxicam, prevented the inhibitory effect of arthritis on body weight gain. Indomethacin and meloxicam administration to arthritic rats increased pituitary GH and liver IGF-I mRNA as well as serum levels of IGF-I. These data suggest that induction of COX-2 during chronic inflammation is involved in the inhibition of the GH-IGF-I axis and in the body weight loss. In the gastrocnemius muscle, arthritis increased the gene expression of tumor necrosis factor (TNF)-alpha, the E3 ubiquitin-ligating enzymes MAFbx and MuRF1, as well as of IGF-I and IGF-binding protein-5 (IGFBP-5). Inhibition of COX-2 by meloxicam administration increased gastrocnemius weight and decreased MAFbx, MuRF1, TNF-alpha, and IGFBP-5 gene expression. In summary, our data indicate that chronic arthritis-induced cachexia and muscle wasting are mediated by the COX-2 pathway resulting in a decreased GH-IGF-I secretion and increased expression of MAFbx and MuRF1 mRNA.

Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis.

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Deltaomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Deltaomp25d mutant were found, especially considering that the fully virulent Deltaomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies.

Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats.

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.

Ghrelin receptor agonist GHRP-2 prevents arthritis-induced increase in E3 ubiquitin-ligating enzymes MuRF1 and MAFbx gene expression in skeletal muscle.

Chronic arthritis is a catabolic state associated with an inhibition of the IGF system and a decrease in body weight. Cachexia and muscular wasting is secondary to protein degradation by the ubiquitin-proteasome pathway. The aim of this work was to analyze the effect of adjuvant-induced arthritis on the muscle-specific ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx) as well as on IGF-I and IGF-binding protein-5 (IGFBP-5) gene expression in the skeletal muscle. We also studied whether the synthetic ghrelin receptor agonist, growth hormone releasing peptide-2 (GHRP-2), was able to prevent arthritis-induced changes in the skeletal muscle. Arthritis induced an increase in MuRF1, MAFbx (P < 0.01), and tumor necrosis factor (TNF)-alpha mRNA (P < 0.05) in the skeletal muscle. Arthritis decreased the serum IGF-I and its gene expression in the liver (P < 0.01), whereas it increased IGF-I and IGFBP-5 gene expression in the skeletal muscle (P < 0.01). Administration of GHRP-2 for 8 days prevented the arthritis-induced increase in muscular MuRF1, MAFbx, and TNF-alpha gene expression. GHRP-2 treatment increased the serum concentrations of IGF-I and the IGF-I mRNA in the liver and in the cardiac muscle and decreased muscular IGFBP-5 mRNA both in control and in arthritic rats (P < 0.05). GHRP-2 treatment increased muscular IGF-I mRNA in control rats (P < 0.01), but it did not modify the muscular IGF-I gene expression in arthritic rats. These data indicate that arthritis induces an increase in the activity of the ubiquitin-proteasome proteolytic pathway that is prevented by GHRP-2 administration. The parallel changes in muscular IGFBP-5 and TNF-alpha gene expression with the ubiquitin ligases suggest that they can participate in skeletal muscle alterations during chronic arthritis.