Dilution Step Research Articles

Metabolomics for determination of non-substance related sleepiness

To date, there is no way to prove non-substance related sleepiness in a forensic context (e.g., after a traffic accident) although it is estimated that drowsy drivers cause 10-20% of all driving accidents. However, the legal situation is clear: the legislator prohibits overtired people from driving a vehicle in road traffic. An objective test for determination of non-substance related sleepiness is desirable. Metabolomics can offer a direct readout of one's phenotype. Changes in physiological functions (such as sleep-wake regulation) are reflected in endogenous metabolism and should therefore be detectable as changes in the metabolic profile, as an increase or decrease of specific metabolites correlating with hours of wakefulness. Our study aimed to assess the feasibility of discovering significant changes in the metabolome associated with hours of wakefulness. Saliva as easy-to-access samples should be used for these studies. Oral fluid samples of 13 volunteers were collected during a previous controlled sleep study that involved a sleep deprivation session and a control session in a randomized cross-over design. After a baseline night of 8 hours sleep, subjects were kept awake for 40 hours in the sleep deprivation session, whereas a 16/8 hours sleep scheme was applied during the control session. Six samples per participant were taken using Salivette® cotton swabs at defined time points across each study session during wakefulness after 0, 16, 25, 31, 38 and 40 hours, respectively. A seventh sample was taken after a recovery night that consisted of 8 hours sleep. Samples were processed using protein precipitation and dilution steps with acetonitrile and analyzed by UPLC-HRMS/MS (Sciex QTOF 6600, IDA) in an untargeted metabolomics setup. Both a reversed-phase (RP) and a hydrophilic liquid interaction (HILIC) column were used and both positive and negative ionization mode were measured to cover a wide range of metabolites. Our analyses detected numerous salivary metabolites in the presented setup (RP: 3845, HILIC: 2434), and many that underwent noticeable changes across the study time course. On the one hand, there are metabolites that increased or decreased in linear manner by hours of wakefulness during sleep deprivation but did not during control session with regular sleep regime. More importantly, some receded to baseline after recovery sleep. On the other hand, significant differences in metabolic profiles were apparent between sleep deprivation and control session. However, high inter-individual differences between study subjects impeded formulation of general statements. In addition to that, there were metabolites that followed a diurnal rhythm (i.e., circadian rhythm), like cortisol, arginine and tyrosine. We have shown that our untargeted metabolomics platform is in principle capable of analyzing salivary metabolites in sleep deprived and control subjects across a time course. Oral fluid is a rich and easily accessible matrix. Due to its simple and non-invasive sampling protocol, it is tailor-made for roadside testing and thus for forensic purposes. While rhythmically changing metabolites are not suitable to serve as marker substances for sleepiness due to their natural swings, those that show linear dynamics correlating with hours of wakefulness may serve as biomarkers. Nevertheless, identification of these remains tedious. However, several further considerations need to be addressed for a future optimized study. If not receding to baseline after recovery sleep, a metabolite cannot be related to sleepiness. High inter-individual differences come into play, too. Also, different sleep habits and therefore different sleep phenotypes are prevalent, resulting in time-shifted endogenous cycles that must be considered. Thus, a strict study protocol is needed for investigation of potential metabolic biomarkers for sleepiness to mitigate or eliminate confounding factors.

Efficient Continuous-Flow Synthesis of Methyl Ethyl Ketone Peroxide in a Microreaction System

Organic peroxides are important to the fine chemical industry because of their applications in polymerization and curing initiators. However, as a hazardous process, the peroxidation reaction to synthesize organic peroxide involves explosive intermediates, sensitive products, and exothermic reactions, resulting in many accidents reported each year. Recently, continuous-flow microreaction synthesis has emerged as a new technology for producing chemicals efficiently and safely. This work introduced a novel efficient continuous-flow microreaction system to intensify the methyl ethyl ketone peroxide (MEKPO) production process. A microreaction platform for MEKPO synthesis was established, with quantitative methods to uncover the effects of reaction conditions on the kinetics and thermodynamics of the reaction, which had never been reported before. Then, a continuous-flow microreaction process was developed to replace the existing batch method by optimizing the peroxidation reaction, phase separation, and dilution steps. Compared to the current batch process for the commercial MEKPO product V688, the conversion of MEK and H2O2 reached 69.11% (4% higher than V688) and 70.01%, respectively, when the nitric acid concentration in H2O2 was 0.5 wt % and the reaction reach time was 270 s at a temperature of 25 °C. Also, our product can achieve the same performance (gel time and gel to cure time) as V688 with a lower active oxygen content (9.5% for V688, 8–9% for microreaction products). These results indicated that both the safety of the peroxidation reaction process and the activity of products were improved by replacing the existing batch process with our continuous microreaction process, which provides great potential for the industrial scale-up of such a system.

Construction of a Porcine Reproductive and Respiratory Syndrome Virus with Nanoluc Luciferase Reporter: a Stable and Highly Efficient Tool for Viral Quantification Both In Vitro and In Vivo.

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for the global pork industry, characterized for its genetic variation and unsatisfied heterological protection from vaccines. A high-throughput screening platform for developing anti-PRRSV therapies is urgently needed. Here, an 11-amino-acid subunit HiBiT derived from NanoLuc luciferase was inserted into the PRRSV genome at four loci of the Nsp2 coding region or as an additional TRS2 driven open reading frame (ORF) between the ORF7 and 3′-untranscribed region (3′-UTR), respectively, and five recombinant viruses with luciferase activity were successfully rescued. The virological characteristics of the representative virus RvJX-Nsp2325-HiBiT were investigated. In vitro, it displayed similar growth kinetics as the parental virus and keeps the luciferase activity and genetic stability after eight rounds of serial passages. The concept-proof test confirmed that RvJX-Nsp2325-HiBiT can be easily used to evaluate the efficacy of antiviral reagents by detecting the reduction of luciferase activity, showing a consistent trend with infectious titers, as well as to set a novel convenient virus neutralization assay based on the intensity of luciferase activity. Last, the viral proliferation, virulence, validity, and HiBiT stability were further investigated in pig inoculation study, showing that the luciferase activity can be directly detected in the tissue samples or indirectly from the MARC-145 cells inoculated with sera from RvJX-Nsp2325-HiBiT-inoculated pigs. Taken together, the results indicate that the HiBiT-tagged virus is a convenient and stable tool for evaluating viral propagation both in vitro and in vivo, which can provide a high-efficient platform for screening and evaluating anti-PRRSV therapies.IMPORTANCE Luciferase reporter tagged virus is crucial to viral quantification in the study of viral replication, pathogenesis and exploring antiviral reagents. It is urgently needed for PRRSV academia to construct a stable, fast, and high-throughput reporting system, which can be used both in vitro and in vivo. Here, an 11-amino-acid luciferase subunit was successfully inserted into the PRRSV genome; the feasibility, genetic stability, and efficiency for viral quantification both in vitro and in vivo were characterized; and the results demonstrated it has greatly improved the convenience and efficiency for screening the anti-PRRSV reagents. Furthermore, a novel luciferase-based virus neutralization assay was successfully set, which can eliminate the step of sample gradient dilution and greatly improve the convenience and throughput of neutralizing antibody testing. Predictably, it will greatly facilitate the screening and evaluating anti-PRRSV therapies, as well as the mechanistic study of its replication and pathogenesis in the future.

Tolerance of the mRNA COVID-19 vaccines in patients with reported taxane reactions

Tolerance of the mRNA COVID-19 vaccines in patients with reported taxane reactions

Label-free study of intracellular glycogen level in metformin and resveratrol-treated insulin-resistant HepG2 by live-cell FTIR spectroscopy

Conventional in vitro study often involves the destruction of the cells followed by purification and dilution steps before applying enzymatic assay or metabolomic analysis. It is a costly and laborious process, and it cannot monitor changes as a function of time. Recently, we have developed a new label-free live-cell FTIR approach that can directly measure biochemical compositional changes within living cells in situ and the spectral changes are shown to be highly specific to the drug applied. In this work, we have demonstrated for the first time the effect of two anti-diabetic drugs, metformin and Resveratrol, on insulin-resistant liver cells (HepG2). Using live-cell FTIR with principal component analysis, we have shown the differences in the biochemical profiles between normal and insulin-resistant cells (p < 0.05), the lack of response/difference from the insulin-resistant cell to insulin (p > 0.05) and the restoration of the biochemical profile and sensitivity to insulin from the insulin-resistant cells after the drug treatment (p < 0.05). Particularly, a rise in the glycogen level, marked by three distinctive peaks at 1150, 1080 and 1020 cm−1, within the living cells after the anti-diabetic drug treatments is observed. The live-cell FTIR results are confirmed by a parallel gold-standard biochemical assay, demonstrating the restoration of insulin sensitivity of the insulin-resistance cells. Live-cell FTIR can be a complementary tool for drug efficacy screening, especially for insulin sensitizers.

Investigation on the Beneficiation of Fluorite-ore using a mixture of Oleic acid and Palmitic acid via Froth Flotation

Fluorite (fluorspar), an important industrial mineral ore, is one of the major raw materials for the production of hydrofluoric acid. Naturally occurring fluorite ore contains gangue minerals, primarily silica (SiO2) and calcite (CaCO3). The beneficiation of fluorspar is needed to mitigate the severity of operational issues. Several studies on fluorspar beneficiation using the froth floatation technique have been executed, but the process is restricted by the use of minerals acids to remove calcite. In this study, a novel scheme for fluorspar beneficiation was investigated for the floatation of fluorite and depression of calcite and other gangue minerals simultaneously without the use of mineral acids. Moreover, mixtures of saturated and unsaturated fatty acids (oleic acid and palmitic acid) were used instead of mineral acids. Various parameters were investigated, such as the weight percent of the feed-slurry, feed pH, the effect of reagents on the process selectivity, and the effect of feed boiling and dilution on the up-gradation of fluorspar. The adopted methodology with tested reagents resulted a 67 wt. % reduction of CO32- and 75 wt. % reduction of SiO2 in gangue material at a pH 10 for the feed slurry. Fluorspar purity increased from 65-75 wt. % to 95 wt. % after the boiling and dilution steps, respectively.

Chemical Characterization in the Production Chain of Permanent Magnets by Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) – Precise Quantification of Nd, Pr, Fe and B in Super-Magnets Samples

Super-magnets, materials whose strong magnetic activity is an attractive differential for the high-tech industry, may have their magnetic performance affected by small variations in their chemical composition. For example, the neodymium and praseodymium content can change the physicochemical properties of the permanent magnets. Aiming at a strict chemical quality control, this work developed an analytical method to quantify the major elements in the materials involved in the production process of didymium (the mixture of neodymium and praseodymium) super-magnets. The simultaneous determination of Nd (401.225 nm), Pr (414.311 nm), Fe (259.837 nm) and B (249.678 nm) in three different sample types (didymium oxide, metallic didymium and (Nd,Pr)-Fe-B alloy) was performed by sample dissolution in acidic media, followed by instrumental measurements using an Inductively Coupled Plasma Optical Emission Spectrometer. Linear calibration curves were obtained with high coefficient of determination (0.9983 ≤ R2 ≤ 0.9999) and with appropriate limits for determining these elements at the percentage level, reaching detection limits less than 0.07 cg g-1. The precision of the method was improved by weighing of the solutions during all the dilution steps and was evaluated by the coefficient of variation associated to instrumental precision (0.3 – 0.7%), method intermediate precision (1.9 – 3.1%) and also by the typical mass fraction provided as uncertainty (0.04 – 0.20 cg g-1), reaching the pressing need to distinguish the content of the rare earth elements in less than 1 cg g-1. The accuracy of the method was assessed by spiked and recovery test (96-104% for spikes equal to or greater than 0.50 cg g-1) and also by the use of different analytical methods, involving the participation of other laboratories, obtaining an acceptable degree of agreement (85 – 107%).

The analysis of volcanic brines by freezing stage tandem LIBS-ICP-MS

Volcanic crater lakes pose unique hazards to the local population both during eruption and quiescence. The composition of crater lake fluids is therefore commonly monitored to aid hazard prediction and mitigation. Some of these fluids, such as at Kawah Ijen in Indonesia and Poas in Costa Rica, are hyper-acidic, concentrated brines (SO4–5 wt%, Cl - 2.5 wt%, Al - 5000 ppm). Analysis of such fluids requires extreme dilution due to the high total dissolved element load, which risks contamination and precipitate formation. To circumvent this undesired sample preparation and speed up the turn-around time on the analysis, we tested approaches which involved freezing the fluids to −30 °C and analysing them by simultaneous Laser Ablation-Inductively Coupled Plasma Quadrupole Mass Spectrometry (LA-ICP-QMS) and Laser-Induced Breakdown Spectroscopy (LIBS). LIBS is highly matrix sensitive, requiring matrix-matched reference materials. At the same time, LA-ICP-MS owes its success to being relatively insensitive to the matrix after accounting for differences in ablation yield. Analysing the fluids as a solid and sampling them by laser circumvents the usual dilution step. Fluid reference materials were prepared by adding the elements of interest to one of the crater lake fluid samples at known, but varying concentrations and by stepwise dilution of one of these fluids. Differences in ablation yield, resulting mainly from variations in the coupling of the laser with the sample, were accounted for by adding a known quantity of lithium as an internal standard. Working curves for both ICP-MS and LIBS were obtained for Al, Mg, Fe, Ca, K, Na, S and Cl, yielding relative trueness values for 4 crater lake samples of known composition of Al-7%, Ca-5%, Fe-1%, K < 1%, Mg-4%, Na-5%, S-3%, and Cl-6% for ICP-MS and Al-2%, Ca-9%, Fe-17%, K-10%, Mg-4%, Na < 1%, S-8%, and Cl-9% for LIBS. For these elements, ranging in concentration from around 400 mg/kg to 25 g/kg, LIBS yielded similar trueness results to ICP-MS. LIBS calibration without an internal standard produces acceptable results for Li only, allowing the cryo-LIBS to analyse the major elements while optimising the ICP-MS for trace-element analysis and obtaining a full suite of elements simultaneously without the need for sample dilution.

NMR analysis: potential and challenges as a homeopathic basic research tool

Background: Homeopathic ultra-diluted solutions surpass the threshold of matter dispersion and, consequently, their chemical constitution is similar to inert solvent. Nevertheless, randomized clinical trials have shown that the clinical effects of these homeopathic medicines are superior to placebo1. Nuclear magnetic resonance (NMR) is one of the most promising techniques to detect physicochemical alterations induced by homeopathic procedures2,3. Aims: To investigate T2 NMR relaxation times of Zincum metallicum and lactose dynamized samples. Methodology: Zincum metallicum samples were ground until 6dH using lactose as an excipient. Subsequently, these samples were dynamized with ultrapure water to produce 8dH, 9dH, 10dH, and 11dH. Lactose dynamized samples (6dH-11dH) were used as control. Aliquots of 540µl of each sample were diluted with 60µl of deuterated water (D2O) in 5mm tubes. The analyses were carried out in Bruker Ascend TM 500MHZ spectrometer at 288 K. Results and discussion: The Zincum metallicum and lactose T2 relaxation times were very similar, except for Zincum metallicum 8dH, which presented a value of 1.226 in comparison to 1.036 of lactose 8dH. The following T2 values were registered: 1.287 - 9dH; 1.413 - 10dH; 1.467 - 11dH, and 1.303 - 9dH; 1.400 – 10dH; 1.350 -11dH, for Zincum metallicum and lactose, respectively. The differences detected in 8dH samples are probably due to the presence of lactose in the first dilution step, in which 1 part of the 6dH triturated mixture was diluted in 9 parts of water, to prepare 7dH. Following this homeopathic procedure, 8dH solutions remain around 1% of lactose which could be influenced by the T2 values registered. Conclusion: These preliminary results showed the possibility to apply the NMR technique to evaluate the influence of dynamization in the relaxation parameters. Further studies should be carried out with other potencies and/or other homeopathic substances, in addition to the evaluation of T1 and the T1/T2 parameters, as previously described by other groups.

Abstract #1153940: Specific Detection of TSAb and TBAb in Serum of Suspected AITD Patients using Two Rapid Homogenous Bioassays

Two rapid homogenous bioassays, named as Turbo TSI and Turbo TBI, have been developed to either quantitatively detect Thyroid Stimulating Antibodies (TSAb) or qualitatively detect Thyroid Blocking Antibody (TBAb) in Autoimmune Thyroid Disease (ATID) patient sera. The two bioassays do not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time to 60 minutes. This study was designed to demonstrate the high specificity of the two bioassays in comparison with the TSHR Autoantibody (TRAb) ELISA Kit (Kronus).

Packed in‐tube SPME–LC–MS/MS for fast and straightforward analysis of cannabinoids and metabolites in human urine

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC-MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508µm i.d.×50mm) stainless-steel hardware; each one required only 12mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 24-1 and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8min), whereas the complete IT-SPME-LC-MS/MS method took another 12 min, with a sample throughput of 3 samplesh-1 . The developed method presented adequate precision, accuracy and linearity; R2 values ranged from 0.990 to 0.997, in the range of 10-1000ngml-1 . The lower limits of quantification varied from 10 to 25ngml-1 . Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times.

Simultaneous MEKC-DAD and smart spectrophotometric assays of thiocolchicoside and etoricoxib in challenging concentration ratio mixtures

Abstract Potent muscle relaxant (thiocolchicoside, TCC) and nonsteroidal anti-inflammatory drug (etoricoxib, ETXB) fixed-dose combination is formulated at relatively high 1:15 and 1:7.5 ratios for TCC and ETXB, respectively. Since the minor component (TCC) has lower absorptivity, assay of TCC/ETXB tablets presents an analytical challenge. The current study presents two novel methods: first is a micellar electrokinetic capillary chromatography (MEKC). Background electrolyte is borate buffer (40 mM, pH 9.2) containing 30 mM sodium dodecyl sulfate and methanol (ratio 80:20%, v/v), measured at 210 nm. Second is a direct double A max spectrophotometric method; minor component, TCC, is measured directly at its distant λ max (373 nm), at zero absorption of ETXB. Then, a ten-fold dilution step is carried out to eliminate TCC spectral interference and ETXB is determined at its λ max (282 nm). Both drugs’ concentrations disclose obedient linearities at 2–100 μg·mL−1 in MEKC, versus 3–25 and 40–350 μg·mL−1 for TCC and ETXB, respectively, in spectrophotometry. All ICH validation elements have been fulfilled for the developed methods. MEKC and spectrophotometric assays achieve accuracy, precision, selectivity, and robustness to be recommended for industrial quality control routine analysis of TCC/ETXB pills formulated at cited ratios or even higher.

Smartphone-enabled rapid quantification of microplastics

Developing methods to quickly detect microplastics is critical to assessing the extent of microplastic contamination in the environment. However, current methods to quantify microplastics from environmental samples can take several hours to days and often require access to expensive specialized microscopy instruments. Herein we report a smartphone-based method to rapidly quantify microplastics. The method involves isolating microplastics from soil or water by density separation and vacuum filtration, staining the isolated plastic polymers with Nile Red, and quantifying the strained microplastics as small as 10 µm using a smartphone-based fluorescence microscope with an opti-mechanical attachment. The smartphone-enabled quantification using an algorithm eliminates time-consuming digestion steps and manual counting, thereby enabling quantification of microplastic concentration in environmental samples within 1 h. The method successfully detected a wide range of plastic polymers, but a dilution step was often needed if the samples contained high concentrations of particulates or non-plastic debris to minimize optical overlap or blocking. This method could serve as an initial assessment tool to rapidly quantify microplastics in environments in remote places with limited access to expensive resources and open the possibility to increase the frequency of monitoring microplastic concentration in engineered systems such as wastewater treatment plants.

Graded coronavirus disease 2019 vaccine administration: A safe alternative to vaccine avoidance

Graded coronavirus disease 2019 vaccine administration: A safe alternative to vaccine avoidance

Development of Quality Control Ranges for Biocide Susceptibility Testing.

Every laboratory test needs validation by quality controls. For biocide susceptibility testing (BST), neither quality control (QC) strains nor QC ranges applicable to these strains are currently available. As QC strains, four well-defined laboratory reference strains (Staphylococcus aureus ATCC® 6538, Enterococcus hirae ATCC® 10541, Escherichia coli ATCC® 10536 and Pseudomonas aeruginosa ATCC® 15442), which have been used previously for biocide efficacy testing, were selected. In an interlaboratory trial with eleven participating laboratories, BST QC ranges should be developed for the aforementioned four strains and the four biocides benzalkonium chloride, chlorhexidine, octenidine and polyhexanide. The performance of three different lots of tryptic soy broth was explored using the broth microdilution method and the data were subsequently evaluated using the RangeFinder software. As a result, QC ranges were defined for all reference strain–biocide combinations, except for P. aeruginosa ATCC® 15442 with the two biocides chlorhexidine and polyhexanide. The development of the latter two QC ranges was not possible, due to the limited solubility of the biocides in the test range required for P. aeruginosa ATCC® 15442. The newly developed QC ranges comprise three to five dilution steps. The establishment of QC ranges will contribute to the validation of BST in the future.

Simulation-based evaluation of single pass continuous diafiltration with alternating permeate flow direction

Abstract In the framework of modern bioprocessing continuous ultrafiltration/diafiltration (UF/DF) is getting increasingly popular. However, while continuous UF can be easily implemented using a so-called single pass tangential flow filtration (SPTFF) module, continuous DF requires a more complicated setup including several SPTFF modules and intermittent dilution steps. Recently, we introduced a novel module design for continuous DF allowing simultaneous delivery of fresh buffer while withdrawing the permeate, thus achieving high degrees of buffer exchange within a single unit. In addition, the system allows to cyclically switch the flow direction of DF buffer through the membranes. Those uncommon features, however, also make it more difficult to determine an operation optimum experimentally by means of trial and error. Therefore, here a detailed finite element model of the physical processes within the module is presented, predicting key figures such as the obtained diafiltration efficiency and the resulting pressures. Because within the module all flow channels are filled by a 3D-printed porous grid supporting the membranes from both sides, the modified Brinkman equation was used to simulate the hydrodynamics, while common mass balance differential equations including accumulation, convection, and an anisotropic dispersion term were used for the simulation of concentration profiles of dissolved species. The predicted key figures are in good agreement with experimental results, obtained for feed solutions including up to 50 g/L of protein and being operated with and without switching the flow direction of the diafiltration buffer. A thorough parameter study reveals that the module shows the best performance for unidirectional flow of the diafiltration buffer, reaching diafiltration efficiencies independence to the applied diavolumes which are comparable to the ones of a conventional multi-stage setup using three SPTFF modules. Therefore, the simulation-based evaluation of optimum operation conditions reveals that the new module design has the potential to realize truly continuous diafiltration setups with high efficiency, requiring only one unit and no extra external piping for returning diafiltration in counterflow. Such simplified setups should be especially useful in small, flexible processing plants as they are increasingly demanded in the biopharmaceutical industry.

A Combined Ultrafiltration/Diafiltration Process for the Purification of Oncolytic Measles Virus.

Measles virus (MV) is an important representative of a new class of cancer therapeutics known as oncolytic viruses. However, process intensification for the downstream purification of this fragile product is challenging. We previously found that a mid-range molecular weight cut-off (300 kDa) is optimal for the concentration of MV. Here, we tested continuous and discontinuous diafiltration for the purification of MV prepared in two different media to determine the influence of high and low protein loads. We found that a concentration step before diafiltration improved process economy and MV yield when using either serum-containing or serum-free medium. We also found that discontinuous diafiltration conferred a slight benefit in terms of the permeate flow, reflecting the repetitive dilution steps and the ability to break down parts of the fouling layer on the membrane. In summary, the combined ultrafiltration/diafiltration process is suitable for the purification of MV, resulting in the recovery of ~50% infectious virus particles with a total concentration factor of 8 when using 5 diavolumes of buffer.

Open tubular capillary ion chromatography with online dilution for small ions determination in drinks

This work aims to develop a more robust, easy-to-use, low-pressure, and cost-effective nonsuppressed open tubular capillary ion chromatography (NS-OTIC) approach with online dialysis for small ions determination in drinks. The fabricated device was applied for two selected columns, including poly(butadiene-maleic acid) on a 50-μm bore and AS18 Latex on 25-µm bore fused silica capillaries, for the separation of mixed cations (Na+, K+, Ca2+, Mg2+) and mixed anions (Cl−, Br−, NO2−, NO3−), respectively. High concentrations of ions (up to 100 mM) level can be directly introduced into the NS-OTIC system without an off-line (manual) dilution step. The linear relationship of the peak area and concentration of model ions can be obtained with a resolution > 1.1. The repeatability of the peak area for both OTIC columns was < 6% RSD. Juice and tea samples were successfully analyzed with % recoveries of 77–112 and 90–119 for cation and anion determinations, respectively.

Evaluation of the effects of hesperidin on fresh and frozen-thawed semen quality using two different cryopreservation methods in Simmental bull.

In the industry of bull semen freezing centers, one-step and two-step semen dilution protocols are two standard and well-known methods in semen freezing process. As the freezing/thawing processes cause detrimental effects on sperm function, the addition of antioxidants can improve sperm characteristics. Hesperidin (Hesp) is an antioxidant used as the male reproductive protective agent. Therefore, the aim of this study was to investigate two different dilution methods, as well as to evaluate Hesp supplementation influence on sperm characteristics in fresh and frozen thawed semen. Semen samples were collected from 12 Simmental bulls. Two separate examinations were conducted in, with and without Hesp supplementation groups. Statistical analysis was performed by an independent t-test, Mann Whitny test, MANOVA and ANOVA tests. In comparison to the one and two-step dilution protocols without Hesp supplementation, the two-step dilution showed greater cryoprotective potential. In the Hesp supplemented group, each semen sample was divided into six equal parts for experimental groups (dilution step method/µM of Hesp). In the both one and two step dilution protocols, significant improvements were detected in semen motility parameters by Hesp administration. Also, oxidative stress status was reduced in seminal plasma of Hesp treatment groups. Interestingly, in comparison with Hesp dosage, 1µM was shown to have greater semen cryoprotective potential. In conclusion,

Impedance Spectroscopy applied to the study of high dilutions of Lycopodium clavatum

Introduction: The Impedance spectroscopy [1] is a technique mainly used to characterize the electrical behavior of solids or liquids samples. This particular technique involves placing the sample of material under investigation between two electrodes (capacitor plates), applying an AC voltage and observing the resulting response across the spectrum of impedance by plotting the real part (Z’) as a function of the imaginary part (Z”) of the impedance. Alternatively, graphs of either the real or the imaginary parts of the impedance can be constructed as a function of the applied voltage frequency. Comparative measurements previously carried out by Miranda et al [2]. have demonstrated clear differences between the impedance values of high dilutions of lithium chloride (LiCl) and the corresponding reference water samples (water which has undergone the same dinamization procedures but without the salt). In this paper the results obtained by applying the spectroscopy of impedance technique in high dilutions of Lycopodium clavatum - Lyc (from 15cH to 30 cH), in comparison to the reference waters, will be presented and discussed.&#x0D; &#x0D; Aims: The objective of this work is to measure the impedance components of both high dilutions of Lycopodium clavatum and reference water samples in the frequency range of 100Hz to 13Mhz, using a successful protocol of sample preparation which has already been used before2. Details of the experimental set-up can be found elsewhere[3].&#x0D; &#x0D; Methodology: Thirty samples of Lyc solutions and thirty reference water samples were produced using the same preparation and measuring protocol. Both groups of liquid samples were measured for dynamizations ranging from 1cH to 30cH, in accordance to the Hahnemanian dynamization method and following the practice suggested by the Brazilian Homeopathic Pharmacopeia. The Lyc solutions were specifically compared to the reference water samples in the potencies of 15cH, 18cH, 23cH and 30cH. It is important to highlight here that all the Lyc solutions and the corresponding reference water samples measured were prepared from the same lot of initial distilled water and submitted to the same steps of dilution and succussion protocol3. Typically three impedance measurements were carried out for each investigated solution, starting with the highest potency. The sample holder (capacitor cell) used during the experiment was careful and systematically cleaned after each measurement.&#x0D; &#x0D; Results: The results obtained show that by choosing either the real part (Z’) or the imaginary component (Z”) of the impedance, it is possible to clearly differentiate the Lyc solutions from the corresponding reference water samples, for the potencies 15cH, 18cH and 30cH. For the potency 23cH, however, this difference is not very significant, as it can be observed in Figure 1.&#x0D; &#x0D; Conclusion: Impedance spectroscopy has demonstrated itself to be a powerful and sensitive technique for the physical characterization of Lycopodium clavatum in high dilutions. The differences obtained for the LiCl dynamizations and the corresponding pure water samples are noteworthy. Also, the results exhibit a non-monotonic behavior over the process of dynamization, indicating that the possibility of contamination during the samples manipulation can be ruled out.