Simultaneous assessment of lipid classes and bile acids in human intestinal fluid by solid-phase extraction and HPLC methods

The purpose of the study reported here was to develop a method for the determination of lipid classes in intestinal fluids, including bile acids (BAs). A solid-phase extraction (SPE) method using C18 and silica columns for the separation of BAs, phospholipids (PLs), and neutral lipids (NLs), including free fatty acids, has been developed and validated. Fed-state small intestinal fluid collected from humans was treated with orlistat to inhibit lipolysis and mixed with acetic acid and methanol before SPE to maximize lipid recoveries. BAs, PLs, and NLs were isolated using lipophilic and polar solvents to promote elution from the SPE columns. The different lipid classes were subsequently analyzed using three separately optimized HPLC methods with evaporative light-scattering detectors. High recoveries (>90%) of all lipids evaluated were observed, with low coefficients of variation (<5%). The HPLC methods developed were highly reproducible and allowed baseline separation of nearly all lipid classes investigated. In conclusion, these methods provide a means of lipid class analysis of NLs, PLs, and BAs in human fed-state small intestinal fluid, with potential use in other fluids from the intestinal tract and animals.     

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

Cloud point formation based on mixed micelle in the presence of electrolyte for extraction, preconcentration, and spectrophotometric determination of trace amounts of hydrazine in water and biological samples

A cloud point extraction process using mixed micelle of the anionic surfactant sodium dodecyl sulfate and the nonionic surfactant Triton X-114 to extract hydrazine from aqueous solutions was investigated. The method is based on the condensation reaction of hydrazine with p-(dimethylamino)benzaldehyde, azine formation, and mixed micelle-mediated extraction of azine in the presence of NaCl electrolyte as an inducing phase separation. An azine product was concentrated in surfactant-rich phase after separation. The optimal extraction and reaction conditions (e.g., surfactant, reagent and electrolyte concentrations, and centrifuge time) were studied and the analytical characteristics of the method (e.g., limit of detection, linear range, preconcentration, and improvement factors) were obtained. Linearity was obeyed in the range of 0.50-110ngml(-1) of hydrazine and the detection limit of the method is 0.08ngml(-1). The interference effect of some cations, anions, and organic compounds was also tested. The method was successfully applied to the determination of hydrazine in water and biological samples.     

Capture of genomic DNA on glass microscope slides

It is well known that DNA strands bind to silica surfaces in the presence of high concentrations of chaotropic salts. We developed simple methods to evaluate binding and recovery of DNA on flat glass microscope slides and compared their properties with commercially available silica "spin columns". Surprisingly, genomic DNA was recovered efficiently from untreated glass slides. Binding and elution times from glass slides were optimized in experiments with DNA samples of various sizes and defined buffers. Average DNA recovery from 500 ng of input genomic DNA varied from 25 to 53% for the glass slide protocol. Yields were comparable to those of spin columns. Human serum albumin and plasma components decreased DNA binding to glass several-fold in a concentration-dependent manner. These results support the concept of using flat glass slides as DNA purification surfaces in microfluidic devices for PCR sample preparation.     

A simplified bioprocess for human alpha-fetoprotein production from inclusion bodies

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.     

Do Bees and Hornets Use Acoustic Resonance in Order to Monitor and Coordinate Comb Construction?

We propose that social hornets and bees, who construct large arrays (known as combs) of cells for hatching and brooding their offspring, exploit ultrasonic acoustic resonances in those cells in order to implement the accurate honeycomb structure exhibited by those arrays. This idea is supported by a number of theoretical considerations, including a detailed analysis of the spectrum of lateral acoustic resonances in a cell with circular cross section, considered as an approximation to the actual perfect hexagon shape. It is also supported by the results of some previous measurements of the acoustic spectrum in a nest of Oriental hornets.     

Developments in multiple headspace extraction

This paper reviews new developments in multiple headspace extraction (MHE), especially its combination with two miniaturized extraction techniques, solid-phase microextraction (SPME) and single-drop microextraction (SDME). The combination of the techniques broadens the applicability of SPME and SDME to quantitative determination of analytes in complex liquid and solid matrixes. These new methods offer several advantages over traditional liquid-solid, liquid-liquid and headspace extraction techniques. The potential applications include extraction of volatiles and semivolatiles from environmental and physiological samples and from different polymer products such as medical and biomedical materials, food packaging and building materials. The theoretical principals of the techniques are also briefly reviewed.     

In Vitro Direct Repeats-mediated Deletion During PCR Amplification

While conducting our research on mutations in the human blood platelet glycoprotein Ib-alpha (GPIbalpha) gene, we detected an unusual deletion of 84 bp. This deletion took place in vitro, during PCR and between two direct repeats. It was observed that the deletion could be detected either by the direct sequencing of the PCR product or after the latter's cloning into a plasmid. After observing a series of four sequenced clones from the same individual, we noticed that while three had the same 84-bp deletion, the fourth exhibited a shorter one. We also noted that there were no cases wherein both deleted and undeleted amplicons coexisted and that several point mutations occurred in the sequence surrounding the deletion. Such Taq errors are statistically more frequent in the "deletion prone DNA" than usual. Interestingly, the deletion was observed only in a DNA, which we call here "deletion prone DNA", whose structure might have been particularly reorganized. Indeed, the mung bean nuclease pre-treatment of this DNA prior to PCR prevented the deletion, thus strengthening the hypothesis that an intra-strand hairpin structure was involved in the deletion process. Direct repeats-mediated deletion is well known in vivo but this is the first report of such "in vitro direct repeats deletion".     

Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes

Trophic ecology has benefitted from the use of stable isotopes for the last three decades. However, during the last 10 years, there has been a growing awareness of the isotopic biases associated with some pre-analytical procedures that can seriously hamper the interpretation of food webs. We have assessed the extent of such biases by: (1) reviewing the literature on the topic, and (2) compiling C and N isotopic values of marine invertebrates reported in the literature with the associated sample preparation protocols. The factors considered were: acid-washing, distilled water rinsing (DWR), sample type (whole individuals or pieces of soft tissues), lipid content, and gut contents. Two-level ANOVA revealed overall large and highly significant effects of acidification for both delta(13)C values (up to 0.9 per thousand decrease) and delta(15) N values (up to 2.1 per thousand decrease in whole individual samples, and up to 1.1 per thousand increase in tissue samples). DWR showed a weak overall effect with delta(13)C increments of 0.6 per thousand (for the entire data set) or decrements of 0.7 per thousand in delta(15) N values (for tissue samples). Gut contents showed no overall significant effect, whereas lipid extraction resulted in the greatest biases in both isotopic signatures (delta(13)C, up to -2.0 per thousand in whole individuals; delta(15)N, up to +4.3 per thousand in tissue samples). The study analyzed separately the effects of the various factors in different taxonomic groups and revealed a very high diversity in the extent and direction of the effects. Maxillopoda, Gastropoda, and Polychaeta were the classes that showed the largest isotopic shifts associated with sample preparation. Guidelines for the standardization of sample preparation protocols for isotopic analysis are proposed both for large and small marine invertebrates. Broadly, these guidelines recommend: (1) avoiding both acid washing and DWR, and (2) performing lipid extraction and gut evacuation in most cases.