N-lauroylsarcosine Sodium Salt Research Articles

A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate

This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (~ 109 copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.

Investigating micelle formation in systems with benzethonium chloride and n-lauroyl sarcosine sodium salt: The impact of thermal effects and ethyl lactate concentration using volumetric, statistical, acoustic, and molecular dynamic methods

Surface active agents (SAAs) are versatile molecules that possess the ability to serve as multifunctional ingredients in a wide range of consumer products across various industrial sectors. These molecules can act as wetting and dispersion agents, emulsifiers, foaming and anti-foaming agents, lubricants, and more. The objective of this study was to evaluate the effect of temperature, and ethyl lactate concentration on micelle formation in systems containing benzethonium chloride (BC) and N-lauroyl sarcosine sodium salt (NLSS). The experimental measurements were conducted at three equidistant temperatures i.e., 298.15 K to 318.15 K and pressure, P=0.1 MPa. Several volumetric as well as compressibility parameters including apparent molar properties (Vϕ,Kϕ,S), partial molar properties (Vϕ°,Kϕ,S°), transport properties (ΔtrVϕ°,ΔtrKϕ,S°), hydration number, etc. have been evaluated. A Bayesian two-factor design with uninformative priors was used to analyze the values of Vϕ°,and Kϕ,S°. It was possible to determine that the temperature factors, and the ethyl lactate (EL) addition significantly influence the results for Vϕ° while for Kϕ,S°it is not appreciable at 5 and 10 % in EL concentration when BC and NLSS systems is compared. The simulations of the micellization process show the impact of temperature, particularly in the presence of ethyl lactate, is evident in the structural and dynamic changes observed in the micelles. Specifically, as the temperature rises from 298.15 K to 318.15 K, the self-assembly process becomes more spontaneous and accelerated, leading to larger and more spherical micelles. These alterations in micelle size, shape, and orientation enhance their capacity to effectively solubilize hydrophobic substances. The computational simulations demonstrated that BC molecules exhibit spontaneous aggregation at the interface of polar-nonpolar systems, resulting in the formation of a monolayer. In this monolayer, the heads of the molecules are oriented towards the polar phase, while the hydrophobic tails are in the nonpolar phase. Similarly, the NLSS system exhibits a continuous and gradual process of self-organization over a period of 120 ns, where NLSS molecules naturally come together to create a single layer arrangement at the zigzag-shaped boundary.

Isolation and Detection of Pathological Tau Species in a Tauopathy Mouse Model.

Tau protein in Alzheimer's disease (AD) and tauopathies becomes insoluble due to hyperphosphorylation, conformational alterations, and aggregation. To analyze insoluble tau and pathological tau species, this study employs a methodology that utilizes wild-type and transgenic tau mice (P310S Tau) tissue extraction using 1% Sarkosyl or N-Lauroylsarcosine sodium salt and the radio immunoprecipitation assay (RIPA) buffer. However, the commonly used methods to study the insoluble tau fraction using detergents like Sarkosyl and RIPA require a large amount of homogenate, which can pose challenges when dealing with small tissue samples. Additionally, the study employs immunohistochemistry to visualize and quantify the pathological tau species in the brain tissue of transgenic mice, aiming to identify and analyze pathological tau species such as hyperphosphorylated tau to further our understanding of tauopathies such as Alzheimer's disease.

Designing Nanomedicines for Breast Cancer Therapy.

In 2020, breast cancer became the most diagnosed cancer worldwide. Conventional chemotherapies have major side effects due to their non-specific activities. Alternatively, short interfering RNA(siRNA)-carrying nanoparticles (NPs) have a high potential to overcome this non-specificity. Lipid-substituted polyethyleneimine (PEI) polymers (lipopolymers) have been reported as efficient non-viral carriers of siRNA. This study aims to engineer novel siRNA/lipopolymer nanocomplexes by incorporating anionic additives to obtain gene silencing through siRNA activity with minimal nonspecific toxicity. We first optimized our polyplexes in GFP+ MDA-MB-231 cells to effectively silence the GFP gene. Inclusion of phosphate buffer with pH 8.0 as complex preparation media and N-Lauroylsarcosine Sodium Salt as additive, achieved ~80% silencing with the least amount of undesired cytotoxicity, which was persistent for at least 6 days. The survivin gene was then selected as a target in MDA-MB-231 cells since there is no strong drug (i.e., small organic molecule) for inhibition of its oncogenic activity. The qRT-PCR, flow cytometry analysis and MTT assay revealed >80% silencing, ~95% cell uptake and >70% cell killing by the same formulation. We conclude that our lipopolymer can be further investigated as a lead non-viral carrier for breast cancer gene therapy.

An experimental and theoretical exploration of the impact of co-solvent on the micellization behaviour of ionic surfactants

In current study, a combined experimental as well molecular modeling study on the effect of Ethyl lactate on the self-aggregation behaviour of N-Lauroyl sarcosine sodium salt (NLSS) and benzethonium chloride (BC) was done. It was found that the micellization phenomenon of both surfactants repressed on raising the concentration of the Ethyl lactate. The electrical conductivity measurements were utilized to calculate the CMC values. Moreover, thermodynamic factors were determined by employing temperature dependence of CMC values i.e. XCMC. The outcomes showed that CMC values of both surfactants rise on increasing the co-solvent concentration and it acts as H2O structure breaker. Values of ΔGm° are negative that becomes less negative on upsurging the concentration of co-solvent, which reveals that the micelle formation becomes less favorable at high fractions of Ethyl lactate + H2O. A systematic exploration of the impact of co-solvent on the self-aggregation behaviour is important in various fields like detergency, pharmaceuticals, etc.

Influence of HSA on micellization of NLSS and BC: An experimental-theoretical approach of its binding characteristics

Blood albumins play a very significant role in several biological operations and act as carriers for the transportation and circulation of various endogenous as well as exogenous materials. Experimental as well as computational studies have been carried out in understanding the major binding properties of the system. This investigation explains the interactions of Human serum albumin (HSA) with N-Lauroyl sarcosine sodium salt (NLSS) and benzethonium chloride (BC), which has been explored by means of conductometric, spectroscopic as well as computational studies. Phenomenon of micellization is hindered in several concentrations of protein (HSA) as well as by temperature. Binding ability of HSA with NLSS and BC has been obtained through UV–visible and fluorescence studies. Computational study revealed the presence of seven binding pockets in HSA, out of which the most stable binding pockets for NLSS and BC includes pocket1 and pocket2.

A novel detergent-based decellularization combined with carbodiimide crosslinking for improving anti-calcification of bioprosthetic heart valve

Currently, valve replacement surgery is the only therapy for the end-stage valvular diseases because of the inability of regeneration for diseased heart valves. Bioprosthetic heart valves (BHVs), which are mainly derived from glutaraldehyde (GA) crosslinked porcine aortic heart valves or bovine pericardium, have been widely used in the last decades. However, it is inevitable that calcification and deterioration may occur within 10–15 years, which are still the main challenges for the BHVs in clinic. In this study, N-Lauroylsarcosine sodium salt (SLS) combined with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were utilized to decellularize and crosslink the heart valves instead of GA treatment. The obtained BHVs exhibited excellent extracellular matrix stability and mechanical properties, which were similar with GA treatment. Moreover, the obtained BHVs exhibited better in vitro biocompatibilities than GA treatment. After subcutaneous implantation for 30 d, the obtained BHVs showed mitigated immune response and reduced calcification compare with GA treatment. Therefore, all the above results indicated that the treatment of SLS-based decellularization combined with EDC/NHS crosslink should be a promising method to fabricate BHVs which can be used in clinic in future.

Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF)

A micellar electrokinetic capillary chromatographic method, a variant of capillary electrophoresis, was developed for the detection and measurement of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients under treatment for idiopathic pulmonary fibrosis. To achieve the best separation a 35 mmol/L N-lauroylsarcosine sodium salt surfactant added with a 16 mmol/L sodium 1-heptanesulfonate solution was used as a running buffer. Analytes were eluted in a fairly short time, 7.35 and 9.40 min for pirfenidone and 5-carboxy-pirfenidone, respectively, at 15 °C and with an applied voltage of 30 kV in an 80 cm, 75 µm i.d. uncoated fused-silica capillary. In the range of 6.25–200 µmol/L, calibration curves exhibited acceptable linearity with the coefficient of determination higher than 0.999. The recovery of analytes spiked in samples of plasma was found to be 95–109% and 93–106% for pirfenidone and 5-carboxy-pirfenidone, respectively. Intra- and inter-day precisions were around 5%. LOD and LOQ for pirfenidone and 5-carboxy-pirfenidone were, respectively, 1.0 and 4.0 µmol/L. The method, applied to measure analytes in six idiopathic pulmonary fibrosis male patients, provided mean concentrations of 68.91 ± 26.31 and 36.21 ± 26.39 µmol/L for pirfenidone and 5-carboxy-pirfenidone, respectively.

Potassium Oleate as a Dissolution and Corrosion Inhibitor during Chemical Mechanical Planarization of Chemical Vapor Deposited Co Films for Interconnect Applications

1,2-4 Triazole (TAZ), N-lauroylsarcosine sodium salt (NLS) and potassium oleate (PO) were tested as passivating additives to previously developed H2O2 and citric acid-based silica dispersions for the polishing of chemical vapor deposited Co films for interconnect applications. In comparison to TAZ and NLS, Co corrosion currents are ∼1 to 2 order of magnitude lower (∼1–3 μA cm−2) at pH 7 with PO and post-polish surface quality was much better. The galvanic corrosion currents between Co-Ti and Ti-TiN couples with PO were also very low (∼0.1 μA cm−2), making the previously developed silica and citric acid-based dispersion, now containing PO, suitable for both bulk removal of Co (step I) and for planarizing Co/Ti/TiN structures (step II) with excellent selectivity while stopping on low-k dielectric. Langmuir adsorption isotherm model analysis showed that the standard free energies of adsorption values were ∼−40 kJ/mol for PO suggesting chemisorption. Fourier transform infra-red (FTIR) and attenuated FTIR spectroscopy data show bridging coordination between the carboxylate ligand of PO and the Co surface.

Recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip for equipment-free detection of Cryptosporidium spp. oocysts in dairy cattle feces.

Cryptosporidium is a widespread protozoan parasite that infects a large number of vertebrate animals, resulting in varying degrees of diarrhea or even death. As dairy cattle feces is an important source of Cryptosporidium spp. infection, development of a handy and accurate detection method via its oocysts in dairy cattle feces would be interesting and necessary. We herein developed a quick detecting method using recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip to detect DNA of Cryptosporidium oocysts in dairy cattle feces. The DNA was released by boiled water with 0.1% N-lauroylsarcosine sodium salt (LSS). The established method was proven to be of higher sensitivity than normal polymerase chain reaction (PCR) amplification with the lowest detection of 0.5 oocyst per reaction, and specificity with no cross reactivity to other common protozoan species in the intestine of dairy cattle. The diagnostic method established herein is simple, rapid, and cost-effective, and has potential for further development as a diagnostic kit for the diagnosis of cryptosporidiosis of dairy cattle.

The comparative studies on the ability of anionic surfactants to bind lead(II) ions

Both heavy metals and surfactants are considered as environmental pollutants, however, the unique interfacial properties of surfactants determine their frequent applications in various processes dedicated to the removal of xenobiotics including metal ions from the contaminated water and soil. Therefore the studies on the effect of metal ions on surfactants properties are of great importance to gain insight into the interactions between these chemicals both in natural environment and during purification processes. The aim of this work was to verify the effect of lead(II) ions on the aggregation of the anionic surfactants: sodium dodecyl sulfate (SDS), N-Lauroylsarcosine sodium salt (Sarkosyl) and Sodium dodecylbenzenesulfonate (SDBS) and to compare the ability of these surfactants to bind these metal ions. The experiments involved electrical conductance measurements of surfactant solutions containing lead(II) ions in various concentrations, pH measurements, potentiometric experiments and the flame atomic absorption spectrometry (FAAS). The analysis of the calculated aggregation parameters and association constants as well as FAAS experiments results evidenced that among the studied surfactants SDS is of the highest, while SDBS of the lowest lead(II) ions binding ability. However, all the investigated compounds at the concentrations lower than Critical Micelle Concentration (CMC) form insoluble complexes with these metal ions. The precipitates differ in their stability, namely they dissolve at the excess of surfactant, however, for SDS this process starts at higher surfactant level as in the case of SDBS and Sarkosyl. Moreover, binding of lead(II) ions to the negatively charged surfactant micelles is the most effective in the case of SDS and the weakest for SDBS.

Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus.

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.

Corrosion inhibition by anionic surfactants of AA2198 Li-containing aluminium alloy in chloride solutions

This paper evaluates the inhibiting action of some anionic surfactants towards AA2198 corrosion in NaCl solutions; the effect of surfactant concentration in relation to chloride amount was determined.On separate electrodes, polarization curves were recorded after 1, 24 and 168h immersion in the aggressive media, while EIS technique continuously monitored the alloy corrosion process.In general, these substances stifled both the cathodic and anodic processes and noticeably shifted the pitting potential (breakdown potential, EBR) in the positive direction. The most efficient compounds were N-lauroylsarcosine sodium salt and sodium dodecyl-benzenesulfonate, able to withstand the effects of 0.1M Cl−.

Topical Surfactant-Induced Pruritus: Involvement of Histamine Released from Epidermal Keratinocytes

Surfactants, an important component of cleansers, often cause itch in humans. Topical application of sodium laurate and N-lauroylsarcosine sodium salt to the skin of mice immediately (for 1-1.5 hours) increased scratching, and the former increased scratching again between 2 and 3 hours after application. Thus, we examined the mechanisms of sodium laurate-induced delayed scratching. Sodium laurate (0.1%-10%) increased delayed scratching and skin surface pH in a concentration-dependent manner. N-lauroylsarcosine sodium salt had no effect on these parameters, and sodium hydroxide solution did not increase delayed scratching. Sodium laurate-induced delayed scratching was markedly inhibited by the H(1) histamine receptor antagonist terfenadine, but it was not affected by mast cell deficiency. Sodium laurate application had no effect on the number of total and degranulated mast cells, and did not induce plasma extravasation or the infiltration of inflammatory cells in the skin. Sodium laurate application increased the histamine content of the epidermis, but not that of the dermis, in normal and mast cell-deficient mice. Sodium laurate application increased the ratio of 53-kDa l-histidine decarboxylase (HDC, a key enzyme for histamine production) to 74-kDa HDC in the mouse epidermis and in a human keratinocyte culture. Sodium laurate increased histamine in the human keratinocyte culture, without affecting cell viability. The present results suggest that sodium laurate induced delayed scratching at an alkaline pH through the increased production of histamine in keratinocytes, which may be due to enhanced processing of 74-kDa to 53-kDa HDC.

N-Lauroyl sarcosine sodium salt mediated formation of hydroxyapatite microspheres via a hydrothermal route

Dandelion-like hydroxyapatite (HA) microspheres were successfully prepared using Ca(NO3)2·4H2O and (NH4)3PO4·3H2O as raw materials and N-Lauroyl sarcosine sodium salt (Sar-Na) as template via a hydrothermal route. The chemical composition, structure, morphology and thermal properties of the samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscope (FTIR), Scanning electron microscope (SEM) and Thermal gravimetric analysis (TG), respectively. The results demonstrate that, Sar-Na has great impact on the morphology of HA. With increasing the amount of Sar-Na, the morphology of HA varies from nanograins to nanorods, finally grows into dandelion-like microstructure. The obtained dandelion-like HA microspheres about 6 μm in diameter are composed of radially oriented nanorods. Furthermore, the possible formation mechanism of morphology change is also discussed.

界面活性剤によって誘発されるかゆみとケラチノサイト-ヒスタミン系の関与

The primary function of surfactants is to remove dirt, exfoliated corneum cells, and microorganisms from the skin. However, the use of toiletries such as soaps and shampoos containing surfactants may cause adverse effects such as cutaneous irritation, dryness, and itching. Recently, skin pathologies, including dry skin, rough skin, and sensitive skin, have increased because of changes in living conditions and lifestyle. Although many people with skin pathologies complain of itching during and/or after skin washing using detergents, the mechanisms of detergent-induced itch are yet to be elucidated. Therefore, in this study, we investigated the mechanisms underlying surfactant-induced itching. We found that topical application of an anionic surfactant sodium laurate at an alkaline pH, but not N-lauroylsarcosine sodium salt at neutral pH, to mouse skin induced scratching, an itch-related response. Additionally, we found that the sodium laurate-induced scratching was inhibited by H(1) histamine receptor antagonist, but not mast cell deficiency. Sodium laurate application increased histamine content and the level of the active form (53 kDa) of L-histidine decarboxylase (HDC) in the mouse epidermis, but not the dermis. Furthermore, addition of sodium laurate to a human epidermal cell culture increased histamine release and HDC levels, without affecting cell viability. These results suggest that surfactants with alkaline properties are pruritogenic and that the pruritus is induced by the histamine released from epidermal keratinocytes. The increase in histamine release may be attributable to the activation of HDC in epidermal keratinocytes.

A simple method for extracting DNA from Cryptosporidium oocysts using the anionic surfactant LSS

Detection of low amounts of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention and control of Cryptosporidium in drinking water supplies as Cryptosporidium causes massive waterborne outbreaks worldwide. As Cryptosporidium has a robust oocyst that is extremely resistant to chlorine and other drinking water disinfectants, both the freeze-thaw method and DNA extraction kits have been commonly used for extracting and purifying DNA from the oocyst. However, the DNA extraction procedures are time consuming and costly. Therefore, a simple and low-cost method to extract and purify DNA from the robust oocyst has been required. In this study, we discussed a simple method for detecting Cryptosporidium DNA with the anionic surfactant, n-lauroylsarcosine sodium salt (LSS) using the loop-mediated isothermal amplification (LAMP) to eliminate the need for the freeze-thaw method and the DNA extraction kits. As a result, Bst DNA polymerase was inhibited by 0.1% LSS but not 0.01% LSS and 5% Triton X-100 or Tween 20. Although DNA was extracted from the oocysts by incubating with 0.1% LSS at 90°C for 15 min, Bst DNA polymerase was inhibited by 0.1% LSS. The inhibition by 0.1% LSS was suppressed by adding 5% of the nonionic surfactants, Triton X-100 or Tween 20. The concentration of LSS in a LAMP tube was 0.01% while that in an incubation tube was 0.1%, because LSS in an incubation tube was diluted by a factor of 10 at the DNA amplification process. Therefore, we found that ten oocysts of Cryptosporidium parvum could be detected by incubation with 0.1% LSS, without removing LSS or adding the nonionic surfactants in the LAMP method.

Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies.

Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.

Evaluation of Extraction Methods for Efficient Detection of Enteric Viruses in Pork Meat Products

We report an in-house protocol for extraction and purification of nucleic acids of enteric viruses, which gives more consistent results than representative commercial methods. The protocol uses 4 M guanidine thiocyanate, 0.5% N-lauroylsarcosine sodium salt, and 25 mM sodium citrate pH 7.0 supplemented with 0.14 M β-mercaptoethanol for lysis of virus particles. The addition of TRIzol followed by chloroform-based separation of the aqueous phase is used to purify nucleic acids from the lysate. RNA precipitation is performed using lithium chloride. This protocol was compared with QIAGEN’s RNeasy Kit and bioMerieux’s NucliSens method, by evaluating the ability of each to detect enteric viruses in a complex food matrix. Three different pork products, i.e., cooked ham, liver, and Spanish fermented sausage (“chorizo”) were artificially contaminated with decreasing numbers of murine norovirus 1 and human adenovirus 2. The extracted and purified viral nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Whereas the two commercial extraction methods did not facilitate robust results (quantification was only possible with some viruses and/or some matrices), when coupled with the in-house protocol the linearity and the efficiency of the quantitative reverse transcription-PCR (qRT-PCR) assays were close to 1 in all the food matrices, independent of the virus. Scalability of the in-house method was evaluated by analysis of 1 and 2.5 g of spiked pig liver samples, and quantification was possible on 1 g samples contaminated with any of the two model viruses. Therefore, the in-house protocol facilitates robust qRT-PCR-based quantitative detection of viruses in pork products, and is moreover relatively cheap and simple to perform.