Decrease of lipid extractability of chloroform-methanol upon water addition to human erythrocytes

The yield of lipids extractable by chloroform-methanol 2:1 from human erythrocytes decreases as a function of the relative amount of water added to--or present in--the erythrocyte pellet prior to the lipid extraction. Only slight modifications are observed as long as the volume of water does not exceed that of the red blood cell pellet. As the volume of added water increases, the phosphatidylserine recovery drops dramatically and tends to zero while the yield of the other phospholipids remains unchanged. This phenomenon is not observed when the lipids are extracted by a mixture of isopropanol-chloroform.     

Tissue Homeostasis in the Wing Disc of Drosophila melanogaster: Immediate Response to Massive Damage during Development

All organisms have developed mechanisms to respond to organ or tissue damage that may appear during development or during the adult life. This process of regeneration is a major long-standing problem in Developmental Biology. We are using the Drosophila melanogaster wing imaginal disc to study the response to major damage inflicted during development. Using the Gal4/UAS/Gal80^TS conditional system, we have induced massive cell killing by forcing activity of the pro-apoptotic gene hid in two major regions of the disc as defined by Gal4 inserts in the genes rotund (rn) and spalt (sal). The procedure ensures that at the end of a 40–48 hrs of ablation period the great majority of the cells of the original Rn or Sal domains have been eliminated. The results indicate that the damage provokes an immediate response aimed to keep the integrity of the epithelium and to repair the region under ablation. This includes an increase in cell proliferation to compensate for the cell loss and the replacement of the dead cells by others from outside of the damaged area. The response is almost contemporaneous with the damage, so that at the end of the ablation period the targeted region is already reconstructed. We find that the proliferative response is largely systemic, as the number of cells in division increases all over the disc. Furthermore, our results indicate that the Dpp and Wg pathways are not specifically involved in the regenerative response, but that activity of the JNK pathway is necessary both inside and outside the ablated domain for its reconstruction.     

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

The Effect of a Physical Activity Program on the Total Number of Primary Care Visits in Inactive Patients: A 15-Month Randomized Controlled Trial

Background

Effective promotion of exercise could result in substantial savings in healthcare cost expenses in terms of direct medical costs, such as the number of medical appointments. However, this is hampered by our limited knowledge of how to achieve sustained increases in physical activity.

Objectives

To assess the effectiveness of a Primary Health Care (PHC) based physical activity program in reducing the total number of visits to the healthcare center among inactive patients, over a 15-month period.

Research Design

Randomized controlled trial.

Subjects

Three hundred and sixty-two (n = 362) inactive patients suffering from at least one chronic condition were included. One hundred and eighty-three patients (n = 183; mean (SD); 68.3 (8.8) years; 118 women) were randomly allocated to the physical activity program (IG). One hundred and seventy-nine patients (n = 179; 67.2 (9.1) years; 106 women) were allocated to the control group (CG). The IG went through a three-month standardized physical activity program led by physical activity specialists and linked to community resources.

Measures

The total number of medical appointments to the PHC, during twelve months before and after the program, was registered. Self-reported health status (SF-12 version 2) was assessed at baseline (month 0), at the end of the intervention (month 3), and at 12 months follow-up after the end of the intervention (month 15).

Results

The IG had a significantly reduced number of visits during the 12 months after the intervention: 14.8 (8.5). The CG remained about the same: 18.2 (11.1) (P = .002).

Conclusions

Our findings indicate that a 3-month physical activity program linked to community resources is a short-duration, effective and sustainable intervention in inactive patients to decrease rates of PHC visits.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00714831","term_id":"NCT00714831"}}NCT00714831     

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria,Cylindrospermopsis and Microcystis

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.     

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 ^-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 ^-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 ^-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4⁺ cells and a reduction in Th17 (CD4⁺IL17⁺) and mature dendritic (MHCII⁺CD11c⁺) cells recruitment. Clodronate depletion of macrophages in Ccr6 ^-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.     

Nosocomial Rhinocerebral Mucormycosis: Two Cases with a Temporal Relationship

Mucormycosis is an unusual fungal infection that usually affects immunosuppressed patients. Small outbreaks of mucormycosis have been previously reported. We present two clinical cases of fatal rhinocerebral mucormycosis with a close temporal relationship between them and a possible nosocomial transmission: case 1 was a 75-year-old male with diabetes and COPD, treated with antibiotics and systemic corticosteroids, who developed rhinocerebral mucormycosis. Case 2 was an 88-year-old woman who was treated with systemic antibiotics and corticosteroids and developed the same infection after insertion of a nasogastric tube. Both patients concurred at the same time in our hospital, and healthcare staff was common to both of them. These cases, along with previously reported cases, highlight that, although infrequent, transmission of the fungus in the hospital environment is a real possibility that should be taken into account in order to initiate contact and air isolation precautions that could avoid nosocomial transmission of this infection.     

Regulation of Coagulation Factor XI Expression by MicroRNAs in the Human Liver

High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3′untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.