Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Positive charges of polyamines protect PSII in isolated thylakoid membranes during photoinhibitory conditions

The effects of the positive charges of amines such as spermine (SPM), putrescine (PUT) and methylamine (MET) on the protection of PSII against excessive illumination were investigated in isolated thylakoid membranes. Under photoinhibition conditions, water oxidation, the kinetics of the Chl fluorescence rise and charge recombination in PSII were affected. A low concentration of SPM (1 mM) added before photoinhibition produced a significant improvement of F(v)/F(0), the oxygen yield and the amplitude of the B-band of thermoluminescence compared with the other amines. Amongst the amines studied, only SPM could protect the photosynthetic apparatus under photoinhibition conditions. This protection was probably provided by the polycationic nature of SPM (four positive charges at physiological pH), which can stabilize surface-exposed proteins of PSII through electrostatic interaction.     

Morphological comparison of the ampullae of Lorenzini of three sympatric benthic rays

This study investigated and compared the morphology of the electrosensory system of three species of benthic rays. Neotrygon trigonoides, Hemitrygon fluviorum and Maculabatis toshi inhabit similar habitats within Moreton Bay, Queensland, Australia. Like all elasmobranchs, they possess the ability to detect weak electrical fields using their ampullae of Lorenzini. Macroscopically, the ampullary organs of all three species are aggregated in three bilaterally paired clusters: the mandibular, hyoid and superficial ophthalmic clusters. The hyoid and superficial ophthalmic clusters of ampullae arise from both dorsal and ventral ampullary pores. The dorsal pores are typically larger than the ventral pores in all three species, except for the posterior ventral pores of the hyoid grouping. Ampullary canals arising from the hyoid cluster possessed a quasi‐sinusoidal shape, but otherwise appeared similar to the canals described for other elasmobranchs. Ultrastructure of the ampullae of Lorenzini of the three species was studied using a combination of light, confocal and electron microscopy. All possess ampullae of the alveolar type. In N. trigonoides and M. toshi, each ampullary canal terminates in three to five sensory chambers, each comprising several alveoli lined with receptor and supportive cells and eight to 11 sensory chambers in H. fluviorum. Receptor cells of all three species possess a similar organization to those of other elasmobranchs and were enveloped by large, apically nucleated supportive cells protruding well into the alveolar sacs. The luminally extended chassis of supportive cells protruding dramatically into the ampullary lumen had not previously been documented for any elasmobranch species.     

The loss of culturability by Escherichia coli cells in seawater depends on availability of phosphate ions and phosphate transport systems

Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mm), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater. Offprint requests to: M.J. Gauthier     

Pioglitazone Improves Fat Distribution,the Adipokine Profile and Hepatic Insulin Sensitivity in Non-Diabetic End-Stage Renal Disease Subjects on Maintenance Dialysis: A Randomized Cross-Over Pilot Study

Background

Fat redistribution, increased inflammation and insulin resistance are prevalent in non-diabetic subjects treated with maintenance dialysis. The aim of this study was to test whether pioglitazone, a powerful insulin sensitizer, alters body fat distribution and adipokine secretion in these subjects and whether it is associated with improved insulin sensitivity.

Trial Design

This was a double blind cross-over study with 16 weeks of pioglitazone 45 mg vs placebo involving 12 subjects.

Methods

At the end of each phase, body composition (anthropometric measurements, dual energy X-ray absorptometry (DEXA), abdominal CT), hepatic and muscle insulin sensitivity (2-step hyperinsulinemic euglycemic clamp with 2H2-glucose) were measured and fasting blood adipokines and cardiometabolic risk markers were monitored.

Results

Four months treatment with pioglitazone had no effect on total body weight or total fat but decreased the visceral/sub-cutaneous adipose tissue ratio by 16% and decreased the leptin/adiponectin (L/A) ratio from 3.63×10⁻³ to 0.76×10⁻³. This was associated with a 20% increase in hepatic insulin sensitivity without changes in muscle insulin sensitivity, a 12% increase in HDL cholesterol and a 50% decrease in CRP.

Conclusions/Limitations

Pioglitazone significantly changes the visceral-subcutaneous fat distribution and plasma L/A ratio in non diabetic subjects on maintenance dialysis. This was associated with improved hepatic insulin sensitivity and a reduction of cardio-metabolic risk markers. Whether these effects may improve the outcome of non diabetic end-stage renal disease subjects on maintenance dialysis still needs further evaluation.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT01253928","term_id":"NCT01253928"}}NCT01253928     

Examination of the Combined Effects of Chondroitinase ABC,Growth Factors and Locomotor Training following Compressive Spinal Cord Injury on Neuroanatomical Plasticity and Kinematics

While several cellular and pharmacological treatments have been evaluated following spinal cord injury (SCI) in animal models, it is increasingly recognized that approaches to address the glial scar, including the use of chondroitinase ABC (ChABC), can facilitate neuroanatomical plasticity. Moreover, increasing evidence suggests that combinatorial strategies are key to unlocking the plasticity that is enabled by ChABC. Given this, we evaluated the anatomical and functional consequences of ChABC in a combinatorial approach that also included growth factor (EGF, FGF2 and PDGF-AA) treatments and daily treadmill training on the recovery of hindlimb locomotion in rats with mid thoracic clip compression SCI. Using quantitative neuroanatomical and kinematic assessments, we demonstrate that the combined therapy significantly enhanced the neuroanatomical plasticity of major descending spinal tracts such as corticospinal and serotonergic-spinal pathways. Additionally, the pharmacological treatment attenuated chronic astrogliosis and inflammation at and adjacent to the lesion with the modest synergistic effects of treadmill training. We also observed a trend for earlier recovery of locomotion accompanied by an improvement of the overall angular excursions in rats treated with ChABC and growth factors in the first 4 weeks after SCI. At the end of the 7-week recovery period, rats from all groups exhibited an impressive spontaneous recovery of the kinematic parameters during locomotion on treadmill. However, although the combinatorial treatment led to clear chronic neuroanatomical plasticity, these structural changes did not translate to an additional long-term improvement of locomotor parameters studied including hindlimb-forelimb coupling. These findings demonstrate the beneficial effects of combined ChABC, growth factors and locomotor training on the plasticity of the injured spinal cord and the potential to induce earlier neurobehavioral recovery. However, additional approaches such as stem cell therapies or a more adapted treadmill training protocol may be required to optimize this repair strategy in order to induce sustained functional locomotor improvement.     

Microbial population dynamics on Golden Delicious apples from bud to harvest and effect of fungicide applications

The microbial population dynamics on apples cv. Golden Delicious were analysed every 15 days between bud and harvest in a fully replicated experiment in northern Spain in 1994 and 1995. The total microbial populations varied with developmental stage, and with prevailing climatic conditions. The predominant mycroflora were the filamentous fungi Cladosporium and Alternaria spp. and white and pink yeasts. Other genera isolated included mainly species of Epicoccum, Fusarium and Acremonium. However, the most important post-harvest pathogens Penicillium expansum and Botrytis cinerea were seldom isolated from ripening apples. Maximum total filamentous fungal populations occurred after fruit set and during early ripening [2 × 10⁴cfu (colony-forming units) g^-1 approximately] while those of bacteria were maximum at bud stage (3.5 × 10⁵and 3.0 × 10⁴ cfu g^-1 in 1994 and 1995 respectively). White yeasts were more numerous than pink yeasts. Endophytic infection of apple buds by Alternaria spp., responsible for core rot, was found in almost all bud tissue. By contrast, Cladosporium spp. were initially isolated later from 12.5–50% of tissue samples during blooming and fruit set. The impact of a four-spray fungicide regime during apple development significantly decreased the total filamentous fungal populations in both years, and that of Cladosporium spp. in 1994. However, bacterial populations were often higher on apples from fungicide-treated plots. Fungicide sprays decreased populations of Cladosporium, Alternaria and white yeasts for a maximum of up to 15–30 days after application. Fungicide application had little effect on endophytic infection of apples by Alternaria spp. between bud and harvest.     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.