<Emphasis Type="Italic">Rhizobium</Emphasis> as plant probiotic for strawberry production under microcosm conditions

There is increasing interest in the use of plant growth-promoting rhizobacteria (PGPR) as environmental-friendly and healthy biofertilizers. Strawberries (Fragraria x ananassa) are mainly consumed fresh and hence any PGPRs used for biofertilization must be safe for humans, which is the case for members of the genus Rhizobium. In this study, the effects of inoculation of strawberry plants with Rhizobium sp. strain PEPV16, which belongs to the phylogenetic group of R. leguminosarum, and whose plant growth promotion ability has been reported previously for lettuce (Lactuca sativa) and carrots (Daucus carota), was examined. The results demonstrated that PEPV16 promotes strawberry growth through significant increases in the number of stolons, flowers and fruits as compared with uninoculated controls. Compared to uninoculated controls, the fruits of the inoculated plants had higher concentrations of Fe, Zn, Mn and Mo, and they also had higher concentrations of organic acids, such as citric and malic acid, and lower amounts of ascorbic acid than fruits. Although decreases in ascorbic acid have previously been described after the inoculation of strawberry with strains from different PGPR genera, this is the first study to report increases in organic acids after PGPR inoculation.     

A PNPase Dependent CRISPR System in Listeria

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in “CRISPRology”.     

The Hepatoselective Glucokinase Activator PF-04991532 Ameliorates Hyperglycemia without Causing Hepatic Steatosis in Diabetic Rats

Hyperglycemia resulting from type 2 diabetes mellitus (T2DM) is the main cause of diabetic complications such as retinopathy and neuropathy. A reduction in hyperglycemia has been shown to prevent these associated complications supporting the importance of glucose control. Glucokinase converts glucose to glucose-6-phosphate and determines glucose flux into the β-cells and hepatocytes. Since activation of glucokinase in β-cells is associated with increased risk of hypoglycemia, we hypothesized that selectively activating hepatic glucokinase would reduce fasting and postprandial glucose with minimal risk of hypoglycemia. Previous studies have shown that hepatic glucokinase overexpression is able to restore glucose homeostasis in diabetic models; however, these overexpression experiments have also revealed that excessive increases in hepatic glucokinase activity may also cause hepatosteatosis. Herein we sought to evaluate whether liver specific pharmacological activation of hepatic glucokinase is an effective strategy to reduce hyperglycemia without causing adverse hepatic lipids changes. To test this hypothesis, we evaluated a hepatoselective glucokinase activator, PF-04991532, in Goto-Kakizaki rats. In these studies, PF-04991532 reduced plasma glucose concentrations independent of changes in insulin concentrations in a dose-dependent manner both acutely and after 28 days of sub-chronic treatment. During a hyperglycemic clamp in Goto-Kakizaki rats, the glucose infusion rate was increased approximately 5-fold with PF-04991532. This increase in glucose infusion can be partially attributed to the 60% reduction in endogenous glucose production. While PF-04991532 induced dose-dependent increases in plasma triglyceride concentrations it had no effect on hepatic triglyceride concentrations in Goto-Kakizaki rats. Interestingly, PF-04991532 decreased intracellular AMP concentrations and increased hepatic futile cycling. These data suggest that hepatoselective glucokinase activation may offer glycemic control without inducing hepatic steatosis supporting the evaluation of tissue specific activators in clinical trials.     

Microbiota of Cow’s Milk; Distinguishing Healthy,Sub-Clinically and Clinically Diseased Quarters

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC.     

Characterization of Cultivable Bacteria from Brazilian Sponges

Among 1,236 colony-forming units (CFU) associated with 11 species of marine sponges collected from a Brazilian coast, a total of 100 morphologically different bacterial strains were analyzed. The phylogenetic diversity of the bacterial isolates was assessed by 16S rRNA gene amplification—restriction fragment length polymorphism (RFLP) analysis, using AluI restriction endonuclease. The RFLP fingerprinting resulted in 21 different patterns with good resolution for the identification of the bacterial isolates at the genus level. The genus Bacillus was the most commonly encountered genus, followed by Kocuria. Regarding the relationship between the morphotypes and species of marine sponges, Mycale microsigmatosa presented major diversity, followed by Dragmacidon reticulatum and Polymastia janeirensis. An antibiotic susceptibility profile of the 100 sponge-associated bacterial strains was determined by the disk diffusion method, and we observed a variable resistance profile, with 15 % of the bacteria being multiresistant. In addition, 71 of 100 strains were able to produce biofilm. These 71 strains were divided into 20 strong biofilm producers, 10 moderate biofilm producers, and 41 weak biofilm producers. The plasmid profile of the 100 bacterial strains was analyzed and 38 (38 %) of these samples possessed one or more plasmids. Studies like this are important to increase the information on these associated bacteria found off the coastline of Brazil, a place which has rich biodiversity that is still unknown.     

Acute effects of incremental inspiratory loads on compartmental chest wall volume and predominant activity frequency of inspiratory muscle

AimThis research aims to analyze the acute effect of incremental inspiratory loads on respiratory pattern and on the predominant activity frequency of inspiratory muscle, taking into account differences in gender responses. Optoelectronic Plethysmography was performed during loads in 39 healthy subjects (20 women), placing 89 markers on the thoracic-abdominal wall to obtain total and regional volumes. Surface electromyography (SEMG) was taken simultaneously on the Sternocleidomastoid and Diaphragm muscles, to calculate the predominant muscle activity frequency through wavelet analysis. Inspiratory loads were performed using Threshold® with 2 min of breathing at different levels, ranging from a load of 10 cmH₂O plus 5 cmH₂O to 40 cmH₂O or fatigue.ResultsInspiratory Time increased during loads. Total and compartmental volumes increased with different regions, changing at different loads. These changes in volume occur earlier in women (20 cmH₂O) than in men (30 cmH₂O). The predominant activity frequency of Sternocleidmastoid muscle decreased at 30 cmH₂O, while Diaphragm activity decreased at 40 cmH₂O.ConclusionThe acute effects of incremental inspiratory loads are increases of total and regional volumes and inspiratory time. As for muscle activity, the predominant activity frequency declined in Sternocleidomastoid and Diaphragm muscles, but at different loads. Such respiratory and SEMG patterns and gender differences should be considered when clinical interventions are performed.