Centrifugation Pellet Research Articles

Camellia sinensis Assisted Synthesis of Copper Oxide Nanoparticles (CuONPs) and Assessment of Its Antioxidant Activity and Zebrafish Embryonic Toxicology Evaluation.

- Camellia sinensis,or oolong tea, is a partially fermented version of tea used in Asian countries. The remarkable reduction activity of the tea extract can potentially be used for synthesizing nanoparticles. Recently,Camellia sinensishas gained popularity for the formulation of some metal nanoparticles. Aim To formulate green synthesis of copper oxide nanoparticles (CuONPs) mediated byCamellia sinensis(oolong tea) and assess its cytotoxicity and antioxidant properties. Materials & Methods Oolong tea extract is prepared and added to CuSO4 solution to synthesize CuO nanoparticles (CuONPs). The centrifugation pellet of CuONPs is collected and subjected to DPPH (2,2 - diphenyl -1- picrylhydrazyl hydrate)and H2O2 assays. The cytotoxicity screening is performed using zebrafish embryos. Results The reducing activity of oolong tea successfully synthesizes the copper nanoparticles. High values are obtained in DPPH (63% inhibition at 10µL concentration, 73% inhibition at 20µL, 80% at 30µL, 85% at 40µL and 90% at 50µL concentrations) and H2O2 (50% inhibition at 10µL concentration, 65% at 20µL, 68% at 30µL, 75% at 40µL and 80% at 50µL concentrations) assays. There are no morphological deformities in the zebrafish and no loss of cell viability or delayed hatching at low concentrations (below 4-8 µL), as shown by the viable embryos with no morphological deformities. Conclusion The study has evidenced high antioxidant activity and minimal cytotoxicity of CuO nanoparticles produced usingCamellia sinensis, thus proving it to be a good biomaterial for a wide range of biological applications.

Evaluation of sequential filtration and centrifugation to capture environmental DNA and survey microbial eukaryotic communities in aquatic environments.

Sequential membrane filtration of water samples is commonly used to monitor the diversity of aquatic microbial eukaryotes. This capture method is efficient to focus on specific taxonomic groups within a size fraction, but it is time-consuming. Centrifugation, often used to collect microorganisms from pure culture, could be seen as an alternative to capture microbial eukaryotic communities from environmental samples. Here, we compared the two capture methods to assess diversity and ecological patterns of eukaryotic communities in the Thau lagoon, France. Water samples were taken twice a month over a full year and sequential filtration targeting the picoplankton (0.2-3 μm) and larger organisms (>3 μm) was used in parallel to centrifugation. The microbial eukaryotic community in the samples was described using an environmental DNA approach targeting the V4 region of the 18S rRNA gene. The most abundant divisions in the filtration fractions and the centrifugation pellet were Dinoflagellata, Metazoa, Ochrophyta, Cryptophyta. Chlorophyta were dominant in the centrifugation pellet and the picoplankton fraction but not in the larger fraction. Diversity indices and structuring patterns of the community in the two size fractions and the centrifugation pellet were comparable. Twenty amplicon sequence variants were significantly differentially abundant between the two size fractions and the centrifugation pellet, and their temporal patterns of abundance in the two fractions combined were similar to those obtained with centrifugation. Overall, centrifugation led to similar ecological conclusions as the two filtrated fractions combined, thus making it an attractive time-efficient alternative to sequential filtration.

Culture-independent bacterial cell extraction from fluid milk and oat-based beverage for basic qualitative microscopy

Butterfat and protein complicate attempts to extract bacterial cells from milk by centrifugation for use in basic microscopy. Some types of bacteria preferentially separate into the butterfat layer upon centrifugation and are lost when this layer is discarded, and the action of bacterial protease enzymes can cause milk proteins to precipitate and partition into the centrifugal pellet. Butterfat and precipitated protein remaining in the centrifugal pellet along with the desired bacterial cells can confound the results of differential staining and microscopy. Oat- and other plant-based beverages, which are often manufactured by dairy processors on shared equipment, present similar hurdles to bacterial extraction and microscopic visualization because of the presence of oils, starch granules, and dietary fiber particles in these products. Herein we describe methods for centrifugal separation of bacterial cells for microscopy from unflavored milk, chocolate milk, and oat-based beverage. Cell suspensions prepared through these methods were used for phase-contrast microscopy, Gram staining, and viability staining. These techniques can be used to provide rapid, culture-independent diagnostic information when bacterial cells are expected to be present in high concentrations, as in the event of sporadic product spoilage or mass product spoilage incidents.

Design of a centrifugal acceleration system for high velocity injection of impurity pellet into EAST tokamak

A centrifugal pellet injection system for high velocity impurity pellet injection to control ELMs and research impurity transport has been developed for the Experimental Advanced Superconducting Tokamak (EAST). The system is composed of the dropper, the centrifugal accelerator, vacuum pumping system and the diagnostic unit. The centrifugal accelerator is designed to accelerate the pellet by the rotating arm driven by a KYKY F100 turbomolecular pump with an adjusted rotating velocity from 0 to 42,300 rpm. In order to evaluate the stability of the rotating arm, the stress distribution of the rotating arm at different rotating velocities is analyzed by the stress field simulation, and the result shows the maximum stress of the rotating arm appears at the transition position between the large circular groove and the acceleration groove. By simulation analysis, it is noted that the arm can work steadily at 2500 rad/s (corresponding to about 398 Hz arm rotation frequency) rotating velocity and may be broken at 4000 rad/s (637 Hz). By the bench testing using the Li pellets of 1 mm diameter as injection material, it shows the centrifugal accelerator can work steadily for a long time under the condition that the rotating velocity doesn't exceed 2200 rad/s (350 Hz) and the maximum velocity of the pellet can reach 286 m/s. Based on the results of simulation and bench test, the centrifugal pellet injection system was installed and then injected Li pellets into the plasma steadily and continuously with the rotating velocity <2200 rad/s on EAST.

Efficiency of pelleting of sugar beet seeds in centrifugal pelleting machine

Seed pelleting is an efficient way to improve the sowing quality of seeds. Diverse positive effect of pelleting is mostly noticeable for beet seeds. Increased size and spherical shape of seeds with shell allows to use precision seeding technology, as well as fertilizers and protective agents being part of the shell, increase their sowing potential and finally increase yields. To obtain pelleted seeds, it is required to use a specialized line with a seed pelleting machine being the main equipment. An improved design of centrifugal batch-operated seed pelleting machine has been developed and manufactured in Belarusian State Agricultural Academy. To activate the process of rolling the shell in the mixing chamber of the pelleting machine, it was proposed to use separately installed blades. Based on theoretical and experimental studies, the main structural and technological parameters of the pelleting machine has been determined to ensure the highest quality of pelleted seeds. Seeds of sugar beet of the domestic variety “Polybel” obtained in a favorable climate were selected for experimental studies. Finished mixture based on bentonite clay was selected as a filler of the seed coat. After the coat formation, the insect-fungicidal dressing agent Prestige was applied to the seeds. The seeds of sugar beet of the domestic variety “Polybel” obtained on experimental pelleting machine showed high results in germination and yield. They are comparable to imported sugar beet hybrids. Pelleting of seeds of domestic varieties of sugar beet on the territory of the Republic of Belarus can become a good alternative to imported pelleted seeds, especially considering their cost, which significantly exceeds the cost of untreated seeds.

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.

Intestinal Parasites in the Diabetic Patient

Objective: To evaluate the risk of intestinal parasite infection in diabetic patients compared to healthy people. Materials and Methods: This is a prospective case-type study including 50 healthy consultant patients and 50 diabetic patients at the Avicenna Military Hospital of Marrakech. The study takes place within the Department of Parasitology - Mycology of the Avicenna Military Hospital of Marrakech in collaboration with the endocrinology department of the same hospital. The study subjects are divided into two groups, one group includes patients with DS who were randomly selected from those followed for diabetes registered and one control group selected and matched with the DS Group for age and sex, the duration of study is two months from 01/06/2018 to 01/09/2018. Three samples are included and obtained from patients after the complete explanation of the process for them and then collected at the parasitology laboratory. For each coprological sample, we performed a parasitological examination of the stool in the fresh state and after Lugol staining. A concentration by Bailenger technique and a Ziehl-Neelsen stain modified with a centrifugation pellet was carried out. Results: The study group consisted of 50 diabetic patients (24 men and 26 women) and 50 control subjects (28 men and 22 women) who were not affected. Parasite infection rate was significantly higher in patients with DM 48% than in controls 10% (P &lt;0.001). The distribution of risk factors in the two groups was not different. In our study, the most frequently found species was Blastocystis hominis (21 cases), followed by Entamoeba histolytica / Dispar (2cas) and Entamoeba coli (2cases). Only the level of Blastocystis hominis was significantly different between the two groups 42% for DM and 4% for control patients with P &lt;0.050. Infection with two species was observed in a single patient with DM who had Entamoeba coli and Blastocystis hominis. Conclusion: Diabetic patients are at greater risk of developing parasitic infections than the normal population, and Blastocystis hominis is specifically an important opportunist in the gastrointestinal tract responsible for parasitic infections in diabetic patients.

Phenotypic and Genotypic Characterization of Mycobacteria Isolates from Buruli Ulcer Suspected Patients Reveals the Involvement of Several Mycobacteria in Chronic Skin Lesions

is a cutaneous mycobacterial disease that occurs in tropical countries in sub-Saharan Africa, South-East Asia, Australia and America. The responsible pathogen is Mycobacterium ulcerans. Cote d'Ivoire is the most affected country, with more than 30 endemic health districts reporting a large number of chronic skin lesions. The clinical forms and the severity of ulcers vary from one patient to another. Samples from suspected patients were analyzed by PCR at Pasteur Institute of Cote d'Ivoire, as recommended by WHO. IS2404 sequence was detected in 61% of cases, incriminating M. ulcerans in chronic cutaneous lesions. For the other cases the etiology was not identified, thus raising several questions. Are all reported Buruli ulcer cases really caused by M. ulcerans? Would other mycobacteria be involved in the occurrence of chronic skin lesions considered as Buruli ulcer? BU suspected patients were enrolled in endemic areas of Cote d’Ivoire. Samples were collected from cutaneous lesions and transported to the lab at +4°C in 2 ml of Middlebrook 7H9 medium supplemented by Cetylpiridium chloride. The centrifugation pellet was taken with saline buffer to perform microscopic examination and mycobacteria isolation on Lowenstein-Jensen medium. Biochemical characteristics were described by the nicotinic acid detection according to Konno protocol, by the Nitrates reduction test, by the catalase activity detection at 22° and 68°C and the Wayne's Tween 80 hydrolysis test. Genotypic characteristics were determined by PCR with 1 ml of bacterial suspension targeting the insertion sequences (IS6110, IS2404, and IS2606), the plasmid virulence genes and Miru-VNTR loci (Miru-1, VNTR 6, VNTR 19, ST-1). A total of 47 mycobacterial strains were isolated with 3 different types of colonies whose microscopic examination showed Acid-Alcohol-Resistant Bacilli. 65.9% of isolates expressed biochemical characters in favor of M. ulcerans strains and 6.4% in favor of M. marinum strains. For 29.8% of isolates, the characteristics were related to atypical mycobacterial species. The genotyping targeting the IS6110, IS2606 and IS2404 insertion sequences allowed simultaneous amplification in 53.2% of isolates. IS2404 was amplified in 93.6% of isolates; IS6110 was amplified in 74.5% of isolates and IS2606 was amplified in 70.2% of isolates. Five genotypes were identified corresponding to various species of mycobacteria: genotypes 1 and 2 accounted for 63.8% with all the 3 insertion sequences and biochemical characteristics in favor of M. ulcerans strains; genotype 4 accounted for 6.4% of isolates with insertion sequences and biochemical characteristics in favor of M. marinum strains; The strains of genotypes 3 and 5 expressed molecular and biochemical characters relating to various non-M. ulcerans mycobacteria. Virulence genes were found in 72.3% of isolates corresponding to 90% of M. ulcerans strains and 60.7% of non-M. ulcerans mycobacteria. This study confirmed the involvement of several genotypes of M. ulcerans and other mycobacteria in chronic cutaneous lesions suspected as cases of in Cote d’Ivoire.

Shielding Current Analysis in High-Temperature Superconducting Film and Its Application

An acceleration performance of a pellet injection system by using a superconducting linear acceleration (SLA) has been investigated numerically. To this end, a numerical code of an equivalent circuit model has been developed for analyzing the shielding current density in a high-temperature superconducting (HTS) film. By using the code, the pellet injection system of the SLA has been reproduced numerically. The results of the computations show that as the initial position approaches the origin, the acceleration performance improves. In addition, the velocity increases with the coil current. As a result, the coil current is preferably as large as possible for the acceleration of the HTS film. Consequently, accelerating for 2 s or more, the pellet injection system using the SLA has a speed similar to a pneumatic pellet injection and a centrifuge pellet injection.

Effect of preparation method and storage period on the stability of saliva DNA

Saliva is an attractive source for oral microbial detection and quantification since sampling is non-invasive and rapid. ObjectivesTo determine whether different saliva preparation methods or preservation time periods affect DNA stability. MethodsSaliva samples from 4 healthy adult volunteers were processed to obtain 3 different preparations: whole saliva, and after centrifugation pellet and supernatant. Purified DNA (MasterPure™) from each sample was divided into 4 aliquots, one for immediate analysis and 3 (stored at −80°C) for later analyses after 1 week and 2 and 6 months. DNA concentrations and qPCR based quantities of Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, Fusobacterium nucleatum, Filifactor alocis and Streptococcus mutans were determined. ResultsDNA concentration did not decrease (P>0.05) during the 6-month period in any sample. Mean (SE) DNA concentrations (ng/μl) in whole saliva were 152.2 (51.2) and 147.8 (50) at day 0 and 6 months, respectively. Similarly, the values for pellet were 134.9 (42.5) and 133.6 (42.9), and for supernatant, 11 (1.9) and 8.9 (2.3), the difference being significant (P<0.001) between supernatant and whole saliva or pellet. The quantities of most bacterial species found at day 0 remained stable over the 6-month period in all saliva preparations. In supernatant, species quantities were lower (P<0.05) than in whole saliva or pellet. ConclusionsDNA concentrations were comparable between whole saliva and pellet, suggesting that either of them can be used for DNA-based analyses. Our results also demonstrated that DNA extracted from saliva can be preserved at −80°C for at least 6 months without decrease in DNA concentration.

Prediction and Modulation of Platelet Recovery by Discontinuous Centrifugation of Whole Blood for the Preparation of Pure Platelet-Rich Plasma

The aim of this study was to describe the behavior of the separation of red blood cells (RBCs) by discontinuous centrifugation (DC) of whole blood to modulate and control the platelet recovery in the preparation of pure platelet-rich plasma (P-PRP). P-PRP is a platelet-rich plasma (PRP) in which the white blood cell layer is not included. To achieve this goal, an analytical model was derived that takes into account the packing of RBCs and predicts the behavior of platelet and plasma recovery efficiencies (PtPlRE) based on the volume of whole blood, the hematocrit, and the volume of supernatant, as a function of the operating variables, centrifugal acceleration, and time. The model was derived from the basic equation of DC, which originates from the equilibrium balance of forces on a particle, and included the addition of one factor that corrected the terminal velocity of RBCs and was also correlated to the PtPlRE in the supernatant. This factor was the ratio between the fractional volume concentrations of plasma and RBCs in the centrifugation pellet after centrifugation. The model was validated and the variability of the data was determined using experimental data from 10 healthy donors in the age range of 25–35 years. The predicted behavior for the packing of RBCs and the PtPlRE was consistent with the behavior seen in the experimental data. Thus, the PtPlRE could be modulated and controlled through centrifugal acceleration, time, and hematocrit. Use of this model based on a physical description of events is the first step of a reliable standardization of PRP preparations.

Purification of a NAD(P) Reductase-Like Protein from the Thermogenic Appendix of the Sauromatum guttatum Inflorescence

A NAD(P) reductase-like protein with a molecular mass of 34.146±34Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108-120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40% of RL was found in the cytoplasm. Another 30% was detected in Percoll-purified mitochondria and the rest, about 30% was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.

A Centrifuge-Based Procedure for Suspended Solids Measurements in Lagoon Sludge

<abstract><title><italic>Abstract.</italic></title> Concentration of suspended solids (SS) is an important parameter in wastewater analysis that is used to understand handling characteristics and to evaluate treatment options and efficiencies. Samples from anaerobic lagoon sludge often have high levels of SS that are difficult to measure by standard filtration techniques. A measurement technique developed in the Environmental Analysis Laboratory of the Department of Biological and Agricultural Engineering at North Carolina State University (NCSU) uses centrifugation to separate solids from liquid samples and quantifies dissolved solids (DS) in the supernatant. The value of SS is then calculated as the difference between total solids (TS) and DS. The method is based on the assumption that dissolved material is not affected by centripetal force up to 10,000 × g and therefore partitions equally between the liquid phase and the water remaining in the centrifugal pellet. The method was compared to the standard filtration method and another centrifuge-based method using lagoon sludge with TS concentrations from 61,000 to 100,000 mg L^-1. The NCSU method was the only method that gave the reasonable result of SS less than the corresponding TS level. The variability of the NCSU method was significantly lower than that of the other methods as well.

Context Surrounding Processing Sites Is Crucial in Determining Cleavage Rate of a Subset of Processing Sites in HIV-1 Gag and Gag-Pro-Pol Polyprotein Precursors by Viral Protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.

Establishment of a semi-automated pathogen DNA isolation from whole blood and comparison with commercially available kits

Molecular methods for bacterial pathogen identification are gaining increased importance in routine clinical diagnostic laboratories. Achieving reliable results using DNA based technologies is strongly dependent on pre-analytical processes including isolation of target cells and their DNA of high quality and purity. In this study a fast and semi-automated method was established for bacterial DNA isolation from whole blood samples and compared to different commercially available kits: Looxster, MolYsis kit, SeptiFast DNA isolation method and standard EasyMAG protocol. The newly established, semi-automated method utilises the EasyMAG device combined with pre-processing steps comprising human cell lysis, centrifugation and bacterial pellet resuspension. Quality of DNA was assessed by a universal PCR targeting the 16S rRNA gene and subsequent microarray hybridisation. The DNA extractions were amplified using two different PCR-mastermixes, to allow comparison of a commercial mastermix with a guaranteed bacterial DNA free PCR mastermix. The modified semi-automated EasyMAG protocol and the Looxster kit gave the most sensitive results. After hybridisation a detection limit of 10 1 to 10 2 bacterial cells per mL whole blood was achieved depending on the isolation method and microbial species lysed. Human DNA present in the isolated DNA suspension did not interfere with PCR and did not lead to non-specific hybridisation events.

Proteomics Analysis Reveals Post-Translational Mechanisms for Cold-Induced Metabolic Changes in Arabidopsis

Cold-induced changes of gene expression and metabolism are critical for plants to survive freezing. Largely by changing gene expression, exposure to a period of non-freezing low temperatures increases plant tolerance to freezing-a phenomenon known as cold acclimation. Cold also induces rapid metabolic changes, which provide instant protection before temperature drops below freezing point. The molecular mechanisms for such rapid metabolic responses to cold remain largely unknown. Here, we use two-dimensional difference gel electrophoresis (2-D DIGE) analysis of sub-cellular fractions of Arabidopsis thaliana proteome coupled with spot identification by tandem mass spectrometry to identify early cold-responsive proteins in Arabidopsis. These proteins include four enzymes involved in starch degradation, three HSP100 proteins, several proteins in the tricarboxylic acid cycle, and sucrose metabolism. Upon cold treatment, the Disproportionating Enzyme 2 (DPE2), a cytosolic transglucosidase metabolizing maltose to glucose, increased rapidly in the centrifugation pellet fraction and decreased in the soluble fraction. Consistent with cold-induced inactivation of DPE2 enzymatic activity, the dpe2 mutant showed increased freezing tolerance without affecting the C-repeat binding transcription factor (CBF) transcriptional pathway. These results support a model that cold-induced inactivation of DPE2 leads to rapid accumulation of maltose, which is a cold-induced compatible solute that protects cells from freezing damage. This study provides evidence for a key role of rapid post-translational regulation of carbohydrate metabolic enzymes in plant protection against sudden temperature drop.

Export of microRNAs and microRNA-protective protein by mammalian cells

The discovery of microRNAs (miRNAs) as a new class of regulators of gene expression has triggered an explosion of research activities, but has left many unanswered questions about how this regulation functions and how it is integrated with other regulatory mechanisms. A number of miRNAs have been found to be present in plasma and other body fluids of humans and mice in surprisingly high concentrations. This observation was unexpected in two respects: first, the fact that these molecules are present at all outside the cell at significant concentrations and second, that these molecules appear to be stable outside of the cell. In light of this it has been suggested that the biological function of miRNAs may also extend outside of the cell and mediate cell–cell communication. We report here that after serum deprivation several human cell lines tested promptly export a substantial amount of miRNAs into the culture medium and the export process is largely energy dependent. The exported miRNAs are found both within and outside of the 16.5 and 120 K centrifugation pellets which contain most of the known cell-derived vesicles, the microvesicles and exosomes. We have identified some candidate proteins involved in this system, and one of these proteins may also play a role in protecting extracellular miRNAs from degradation. Our results point to a hitherto unrecognized and uncharacterized miRNA trafficking system in mammalian cells that is consistent with the cell–cell communication hypothesis.

Macro to nano: a simple method for transporting cultured cells from milliliter scale to nanoliter scale

Microfluidic devices are well-suited for the study of metabolism and paracrine and autocrine signaling because they allow steady or intermittent perfusion of biological cells at cell densities that approach those in living tissue. They also enable the study of small populations of rare cells. However, it can be difficult to introduce the cells into a microfluidic device to achieve and control such densities without damaging or clumping the cells. We describe simple procedures that address the problem of efficient introduction of cells and cell culture media into microfluidic devices using small bore polyetheretherketone (PEEK) tubing and Hamilton gastight syringes. Suspension or adherent cells grown in cell culture flasks are centrifuged and extracted directly from the centrifuge pellet into the end of the PEEK tubing by aspiration. The tube end is then coupled to prepunched channels in the polydimethylsiloxane microfluidic device by friction fitting. Controlled depression of the syringe plunger expels the cells into the microfluidic device only seconds following aspiration. The gastight syringes and PEEK tubing with PEEK fittings provide a non-compliant source of pressure and suction with a rapid response time that is well suited for short-term intramicrofluidic cellular studies. The benefits of this method are its simplicity, modest expense, the short preparation time required for loading appropriate numbers of cells and the applicability of the technique to small quantities of rare or expensive cells. This should in turn lead to new applications of microfluidic devices to biology and medicine.

Simple quantification of bacterial envelope-associated extracellular materials

We have developed a simple method for distinguishing between bacterial cultures that produce different amount of exopolysaccharide. It is based upon small differences in pellet volume formed by those cultures upon centrifugation. For that we have constructed a special centrifugation tube consisting of two connected chambers: an upper 12 ml chamber connected to a lower capillary chamber. Cells are applied to the upper chamber and following centrifugation, sink to its bottom and are forced into the capillary so that the height they fill can be measured. This procedure has been developed in order to demonstrate differences in volume of centrifugation pellet formed by similar number of Escherichia coli K12 wild type, rpoS mutant and yjbG rpoS double mutant cells. These differences are further shown to be a result of overproduction of colanic acid exopolysaccharide in the mutant strains. We suggest that this simple method can be employed to detect differences in other cell surface structures and to estimate biomass when optical density measurement or microscopic count is not applicable.

Flocs surface functionality assessment of sonicated activated sludge in relation with physico-chemical properties

Flocs surface functional groups evolutions due to an ultrasonic treatment were investigated in respect with the mechanisms involved during sonication. Activated sludge surface functional groups changes were studied after treatment of a sample at different ultrasonic specific energies. Sludge functionality was qualitatively assessed by recording the infrared (FT-IR) spectra of centrifugation pellets. Potentiometric titration coupled with proton surface complexation modeling was used to assess the nature and quantity of ionizable functional groups present at the floc surface and in the aqueous phase. These evolutions were linked to changes of both mixed liquor biochemical composition (TSS, VSS, COD, proteins, humic like substances, polysaccharides) and physical properties (floc size and settleability). Observations carried out showed that activated sludge flocs were essentially mechanically disintegrated by ultrasonic waves: the nature of chemical bonds observed by FT-IR did not shift after ultrasonic treatment. Moreover, the total number of ionizable functional groups measured by potentiometric titrations remained constant during sonication. However, due to the solubilization of organic components induced by cavitation process, the corresponding ionizable functional groups (carboxyl, hydroxyl, amine) were transferred from particulate to soluble fraction. Moreover, due to the variable amount of proteins, humic like substances and polysaccharides solubilised, the relative contributions of carboxyl, hydroxyl and amine groups varied at floc surface.