Use Of Centrifugation Research Articles

Artificial Gravity Attenuates the Transcriptomic Response to Spaceflight in the Optic Nerve and Retina

The development of eye pathology is a serious concern for astronauts who spend time in deep space. Microgravity is a major component of the spaceflight environment which could have adverse effects on ocular health. The use of centrifugation to exert forces that partially or fully mimic Earth-level gravity in space is a possible countermeasure to mitigate the effects of microgravity on the eye. Therefore, we subjected mice on the International Space Station (ISS) to microgravity (0 G) or artificial gravity by centrifugation at 0.33 G, 0.67 G, and 1 G, and then performed RNA sequencing (RNA-seq) on optic nerve and retinal tissue after returning them to Earth alive. We find that the microgravity environment induces transcriptomic changes in the optic nerve and retina consistent with an increased oxidative stress load, inflammation, apoptosis, and lipid metabolic stress. We also find that adding artificial gravity on board the ISS attenuates the transcriptomic response to microgravity in a dose-dependent manner. Such attenuation may effectively protect from and mitigate spaceflight-induced detrimental effects on ocular tissue.

The Use of Centrifugation for the Separation of Suspended and Colloidal Forms of Chemical Elements in the Analysis of River Waters: Possibilities and Limitations

The Use of Centrifugation for the Separation of Suspended and Colloidal Forms of Chemical Elements in the Analysis of River Waters: Possibilities and Limitations

Simultaneous extraction of polycyclic aromatic hydrocarbons and tetracycline antibiotics from honey samples using dispersive solid phase extraction combined with dispersive liquid-liquid microextraction before their determination with HPLC-DAD

A dispersive solid-phase extraction method combined with dispersive liquid-liquid microextraction was offered for the extraction of four polycyclic aromatic hydrocarbons (anthracene, fluorene, phenanthrene, and pyrene) and tetracycline antibiotics (tetracycline, oxytetracycline, and chlortetracycline) from honey samples, simultaneously. In this work, cobalt-gallic acid bio-metal organic framework was synthesized and 20 mg of it was added into of 10 mL the diluted honey solution to efficiently adsorb the analytes. After vortexing (3 min) and centrifuging, the sorbent particles were eluted with 0.75 mL acetonitrile under vortexing for 3 min. In the following, this phase was mixed with 55 µL [Ch: 4-chlorophenol]+[FeCl4]- magnetic deep eutectic solvent and rapidly injected into deionized water. After separating the extractant phase in the presence of an external magnet and diluting it with the mobile phase, it was injected into a high-performance liquid chromatography-diode array detector. Under optimum extraction conditions, extraction recoveries were in the range of 63–84%. The limits of detection of the opted polycyclic aromatic hydrocarbons and tetracyclines were in the ranges of 0.18–0.26 and 0.25–0.29 ng g−1, respectively. Also, good repeatability (relative standard deviations ≤ 7.5 and 8.3% for intra– (n=6) and inter-day (n=4) precisions, respectively) was obtained. In summary, the validated method was successfully recommended for the determination of polycyclic aromatic hydrocarbons and tetracyclines in various honey samples and pyrene was found in two samples at 5.5 and 12.9 ng g−1. The used bio-metal organic framework has sufficient ability in extraction of different classes of compounds (polycyclic aromatic hydrocarbons and antibiotics) from honey. Performing the microextraction step has direct effect in analysis of the analytes in low concentrations and the use of magnetic solvents, eliminate the use of centrifugation in this step.

Processing stallion semen for on‐farm use and cooled transport

SummarySimple dilution of semen is the oldest method to process semen for artificial insemination with either fresh or cooled semen. This was developed prior to the optimisation of centrifugation techniques for semen processing through a combination of trial and error and research. Simple dilution is a faster method to process semen, is easy to use and works in many circumstances. For on‐farm semen use a 1:1 (v/v) semen to extender ratio is used. A variety of other techniques have been developed, such as cushion centrifugation, density gradient centrifugation and sperm filtration. Cushion centrifugation is used to concentrate the sperm and allows higher centrifugation speeds than centrifugation alone. This allows for improved recovery rates of sperm. Density gradient centrifugation has been used to select for morphologically normal sperm but suffers from low recovery rates. Processing sperm with this technique can result in improved motility and improved DNA integrity of sperm. Sperm filtration is another method to concentrate sperm without the use of centrifugation; however, it has lower recovery rates compared to cushion centrifugation. This review will discuss these techniques in the application of processing equine semen for either fresh or cooled‐shipped use.

A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

Life cycle assessment of sericin recovery from silk degumming wastewaters

Sericin is a silk protein discarded with silk degumming wastewater. Life cycle assessment of sericin recovery from silk degumming wastewater was conducted considering two scenarios; base scenario (no recovery), and sericin recovery with membrane hybrid processes. Base scenario consists of silk degumming and treatment of degumming wastewater. The recovery scenario consists of sericin recovery with the process train; silk degumming, sedimentation, nanofiltration (NF), precipitation and lyophilization. Spray drying was also considered as an alternative to the precipitation and lyophilization. The system boundary was set as cradle to grave and the functional unit was selected as 1 kg of sericin. The recovery scenarios had much higher environmental impacts as compared to the base scenario. In terms of kg CO2 equivalence, the impact of the recovery scenarios on climate change (global warming effect) was found to be approximately 2000–2650 times higher than the base scenario. In recovery scenario, endpoint impact score for climate change was as high as 50%, followed by natural resources (34%) and human health impacts (15%). The use of centrifugation + lyophilization option was found to cause 40%–50% more impacts compared to the spray drying option. The highest impact was for the nonrenewable energy impact category, followed by the global warming category. NF and sericin degumming were the processes with highest environmental impacts.

Continuous electrocoagulation of Chlorella vulgaris in a novel channel-flow reactor: A pilot-scale harvesting study

The most frequently used method to harvest microalgae on an industrial scale is centrifugation, although this has very high energy costs. To reduce these costs, a continuous electrocoagulation process for harvesting Chlorella vulgaris was developed and tested using a pilot-scale 111 L working volume device consisting of an electrolyser with iron electrodes, aggregation channel and lamellar settler. The flow rate of the microalgal suspension through the device was 240 L/h. When using controlled cultivation and subsequent electrocoagulation, a high harvesting efficiency (above 85%), a low Fe contamination in the harvested biomass (<4 mg Fe/g dry biomass, a harvested biomass complied with legislative requirements for food) and significant energy savings were achieved. When comparing electrocoagulation and subsequent centrifugation with the use of centrifugation alone, energy savings were 80 % for a biomass harvesting concentration of 0.23 g/L. Electrocoagulation was thus proven to be a feasible pre-concentration method for harvesting microalgae.

Evaluation of offshore oil spill response waste management strategies: A lifecycle assessment-based framework

This study presents a novel life cycle assessment-based framework for low-impact offshore oil spill response waste (OSRW) management. The framework consists of design of experiment, life cycle assessment (LCA), multi-criteria decision analysis (MCDA), operational cost analysis, and generation of regression models for impact prediction. The framework is applied to four OSRW management strategies as different combinations of solid and liquid oily waste collection, segregation, transportation, and treatment/disposal technologies. Hypothetical scenarios based on oily waste compositions are developed, and the associated environmental impacts and operational costs are evaluated. The LCA results show that oily waste composition accounts for < 5% of the total environmental impacts. Chemical demulsification has the highest total impacts due to high marine ecotoxicity and human toxicity, followed by incineration and transportation. The cost analysis reveals that the strategy comprised of centrifugation and landfilling is most preferable while the combination of chemical demulsification and incineration is least favorable. The strategy of combined use of centrifugation and landfilling is ranked as the most suitable in the MCDA. Regression models are developed to predict environmental impacts based on important factors. The framework can help waste management practitioners select low-impact strategies for handling offshore OSRW.

The use of differential separation and density gradient with AllGrad 90% after thawing improves the sperm quality of South American catfish (Rhamdia quelen)

The use of technologies in assisted reproduction, such as the use of centrifugation, has been reported as a tool that allows separating the best portion of sperm from cryopreserved milt. The main of this work was to compare two separation techniques, differential and density gradient, on thawed Rhamdia quelen milt, based on sperm functionality and fertilization capacity. Three treatments were compared: control, differential centrifugation or pelletization (T1), and density gradient centrifugation (T2). Motility percentage was better for differential (42.58 ± 6.50%) than for the control treatment (32.44 ± 6.03%). Membrane integrity and kinetic parameters was not different between treatments. The presence of normal sperm was higher in T2 treatment (13.55 ± 5.82%) when compared to the control (10.72 ± 3.53%) and T1 (3.97 ± 1.95%). There was a lower rate of morphological tail damage in the T1 (26.91 ± 7.13%) treatment compared to the T2 (37.48 ± 10.81%) and control (45.88 ± 4.84%). For the head morphologies, the lowest percentages were for the control (35.72 ± 5.89%) and T2 (4.89 ± 14.94%) treatments compared to the T1 treatment (62.59 ± 9.67%). In the MTT (3- (4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) test (AU/100 million sptz), the centrifuged treatments resulted in values of 1.58 ± 0.79 (T1) and 2.02 ± 1.88 (T2), better than the treatment control (0.20 ± 0.08). Likewise, the fertilization and hatching rates, respectly, were better for the T1 (76.68 ± 15.22% and 54.62 ± 17.39%) and T2 (69.85 ± 17.85% and 50.87 ± 20.59%) treatments, than the control treatment (40.03 ± 11.67% and 24.14 ± 13.97%). The comparison of differential centrifugation with density gradient centrifugation was carried out for the first time with cryopreserved milt of R. quelen. In summary, both centrifugation techniques proved to be efficient in obtaining a better portion of functional sperm from thawed milt. This is based on the high rates of embryonic survival and hatching obtained after both differential and density gradient separation.

Centrifugal force assessment in ram sperm: identifying species-specific impact

BackgroundCentrifugation is routinely employed in handling the ejaculates of some species, but it is not part of the commonly used protocols in ram. However, the development and implementation of new assisted reproductive technologies, alternative preservation models based on washing sperm from a cellular ageing-accelerating substance such as the seminal plasma, and basic studies in spermatology is associated with the use of centrifugation. This requires a specific evaluation of the centrifugation protocols considering the species-specific relationship with the potential damage produced by this procedure. No previous studies have determined the effect of different centrifugation forces on ram sperm. Therefore, we aimed to assess the performance of three centrifugal forces (600×g, 3000×g, and 6000×g for 10 min at room temperature) and their effects on ram sperm motility and functionality.ResultsSperm motility and functionality parameters were assessed at 0 h and after 2 h of incubation at 37 °C. As expected, a higher cell packaging degree was obtained at high centrifugation forces (P ≤ 0.0001). Cell packaging was unstable at all centrifugal forces. Thus, there was a high cell resuspension rate after less than 2 min. Regarding sperm quality, there was a change in movement pattern of 3000×g and 6000×g centrifuged sperm after 2 h of incubation at 37 °C, characterized by an increase in rapid progressive motility, linearity, straightness, and beat frequency, and a decrease in medium progressive motility, curvilinear velocity, path velocity, and head lateral amplitude. Non-significant differences were obtained among the different treatments concerning the total viability. However, we observed a significant increase (P ≤ 0.05) in the percentage of viable apoptotic sperm in the samples centrifuged at 6000×g at 0 h.ConclusionsCentrifugal forces equal to or greater than 3000×g induced some deleterious effects in ram sperm quality, and lower forces did not provide a successful cell packaging degree.

A New Centrifugation-Free Approach for Reducing IgA Levels in Units of Stored Red Blood Cells

Introduction: Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients when RBCs from IgA-deficient donors are not available. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA to levels suitable for IgA-deficient recipients in a single washing cycle, hence double washing is frequently required. Additionally, cell processors are designed to wash one RBC unit at a time, making washing several units for a multiple-unit transfusion a laborious, lengthy and logistically difficult process. Importantly, these conventional cell washers are complex, expensive machines that may not be available in smaller medical facilities, thus limiting availability of washed RBCs on demand. To address these limitations, we developed a simple, portable, disposable system capable of washing stored RBC units at practical volumetric throughput without the use of centrifugation, while reducing IgA below 0.05 mg/dL in a single run. This new washing system consists of two core modules: (i) a coil of narrow-bore plastic tubing with three sequential bifurcations for high-throughput separation of washed RBCs from washing solution, and (ii) a highly-engineered membrane-hydrogel composite for concentrating the suspension of washed RBCs to the desired level of hematocrit.Materials and Methods: Several units of stored RBCs (n = 4, leukoreduced, 4-6 weeks storage duration) were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Samples of stored RBCs were withdrawn from the units and diluted with 0.9% saline to a hematocrit (Hct) of 10%, to match the dilution typically used in the conventional centrifugation-based washing procedure (2000 mL of saline per typical 350 mL RBC unit with a 60-65% initial Hct). The diluted RBC samples were then washed either by our new washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics.Results and Discussion: Stored RBCs were diluted to 10% Hct with normal saline and passed through the new washing system at 5.0 mL/min to produce washed RBCs with output Hct of about 42%, and Hct of washing solution (waste) of about 2%. Overall, washing performed by either of the two methods substantially reduced hemolysis, lowered the concentration of free hemoglobin (by >4-fold) and total free protein (by >10-fold), while keeping the intracellular ATP unchanged (Table 1). Washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) from 62.9 ± 14.3% in the initial RBC units to 74.1 ± 6.9% for our experimental device, which was a more pronounced improvement than for conventional centrifugation (67.8 ± 5.1%), although the difference was not statistically significant. Importantly, our new washing system reduced the level of IgA to well below 0.05 mg/dL required by AABB in a single pass, significantly outperforming the traditional, centrifugation-based washing procedure.Conclusions: This study demonstrated the feasibility of washing stored RBCs in normal saline for transfusion to patients with selective IgA deficiency without the use of centrifugation. Our inexpensive, portable, disposal system was significantly better than centrifugation in reducing the IgA level, and performed on par with the conventional procedure for all other key in vitro metrics of RBC quality. The ability of this new washing system to operate in line with existing infusion equipment at bedside could be particularly attractive in remote or resource-limited settings, when conventional centrifugation-based cell washers are unavailable.Acknowledgments: This work was supported by the 2012 NIH Director's Transformative Research Award (R01HL117329). [Display omitted] DisclosuresShevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Towards a Closed System for Extracting Lymphocytes from Whole Blood

Introduction: Cellular therapies promise new treatment options and potentially curative efficacy for a wide array of acute and chronic diseases. The ability to obtain viable, uncontaminated lymphocyte concentrates from peripheral blood is in high demand. A major barrier for translating lymphocyte-based therapies into clinical practice is the poor scalability of manual, labor- and time-consuming ‘open system’ methods (e.g. Ficoll density gradient centrifugation) currently being used for isolating lymphocytes. To address the well-known limitations of existing separation methods, we developed a two-stage cell isolation approach that enables highly-efficient extraction of lymphocytes from whole blood (WB) without the use of centrifugation or any open-air manipulations by (1) depleting ~99% of red blood cells (RBCs) from the sample via rapid sedimentation induced by HES (6% hydroxyethyl starch in 0.9% sodium chloride), and then (2) passing the leukocyte-rich supernatant through a high-throughput microfluidic device (based on our newly-developed approach of ‘controlled incremental filtration,‘ CIF) to separate lymphocytes from platelets and residual RBCs.Materials and Methods: WB was collected from healthy consenting volunteers under an IRB approved protocol. An optimization study was performed to identify parameters of the sedimentation protocol with the highest lymphocyte yield and minimal contamination. This optimal protocol was then used to compare performance of (i) the standard Ficoll density gradient separation method, and (ii) our new two-stage separation approach. Each WB sample was divided into two equal parts - one half was processed via the conventional density gradient method, and the other half was first mixed with HES, allowed to sediment, and then the supernatant was passed through a CIF-based microfluidic device (designed for high-precision separation of lymphocytes from smaller cells, and ~15-fold reduction of the sample volume) at a flow rate of 0.5 mL/min. For both separation methods, product yield and purity were quantified by performing CBC with a 5-part differential on all samples. Flow cytometry was used to measure viability of isolated lymphocytes, and activation level of residual platelets via P-selectin and phosphatidylserine (PS) exposure. Separated lymphocytes were cultured for 4 days using standard proliferation techniques. Total number of viable cells was determined using Trypan Blue exclusion dye and manual counting with a hemocytometer.Results and Discussion: The optimized sedimentation protocol, which involved mixing WB with 10% HES (v/v) and waiting 30 min to complete, yielded the best combination of lymphocyte recovery (72.1±2.2%) and RBC depletion (99.48±0.07%) for the first stage of our separation approach. In the split-sample study, our two-stage method overall recovered 70.4±0.7% of lymphocytes from the initial WB sample, with 98.2±0.6% viability, while depleting 92.4±0.4% of platelets and 99.62±0.1% of RBCs. In comparison, the Ficoll density gradient centrifugation method removed 99.95±0.2% of RBCs and 94.2±0.1% of platelets, while recovering 74.5±2.40% of lymphocytes, with 98.3±0.7% viability. Lymphocytes produced by our method showed a significnatly higher prolifiration capacity than cells separated via density gradient centrifugation (Fig. 1). Markers of activation for residual platelets in the lymphocyte concentrate produced by our two-stage method were several fold lower than for Ficoll density gradient separation: 1.9±0.1% (our method) vs. 8.4±7.0% (Ficoll) for P-selectin, and 2.0±1.5% (our method) vs. 4.4±1.5% (Ficoll) for PS exposure. Importantly, lymphocyte isolation using our two-stage method was 3-fold faster than the Ficoll density graduate separation for the same amount of WB sample: <60 min (our method) vs. >180 min (Ficoll).Conclusion: We developed an easy-to-use approach for extracting lymphocytes from peripheral blood with purity, viability and yield meeting the current standard for cellular therapies. Since our new approach is scalable, compatible with ‘closed system’ operation, produces lymphocytes with higher proliferation capacity, and is much faster than conventional density gradient separation, it could have a potentially transformative impact by accelerating research into new cellular therapies, and simplifying translation of these treatments into clinical practice. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Ultracentrifugation Techniques for the Ordering of Nanoparticles.

A centrifugal field can provide an external force for the ordering of nanoparticles. Especially with the knowledge from in-situ characterization by analytical (ultra)centrifugation, nanoparticle ordering can be rationally realized in preparative (ultra)centrifugation. This review summarizes the work back to the 1990s, where intuitive use of centrifugation was achieved for the fabrication of colloidal crystals to the very recent work where analytical (ultra)centrifugation is employed to tailor-make concentration gradients for advanced materials. This review is divided into three main parts. In the introduction part, the history of ordering microbeads in gravity is discussed and with the size of particles reduced to nanometers, a centrifugal field is necessary. In the next part, the research on the ordering of nanoparticles in analytical and preparative centrifugation in recent decades is described. In the last part, the applications of the functional materials, fabricated from centrifugation-induced nanoparticle superstructures are briefly discussed.

Optimized in vitro isolation of different subpopulation of immune cells from peripheral blood and comparative techniques for generation of monocyte-derived macrophages in small ruminants

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.

Development and Application of a Modified Method to Determine the Encapsulation Efficiency of Proteins in Polymer Matrices

A modified method to determine protein encapsulation efficiency in polymer matrices has been developed and applied to two proteins and two polymers to demonstrate its wide range of applicability. This study was pursued due to the wide variation in reported protein encapsulation efficiency of polymer-based microcapsules, even when the protein, the polymer, and the microcapsule manufacturing method were consistent. Hemoglobin (Hb) and bovine serum albumin (BSA) were chosen as model proteins and ethylcellulose and poly(lactic-co-glycolic acid) (PLGA) as model polymers. The polymer of the microcapsule was dissolved in dichloromethane/ethanol or dichloromethane/ethyl acetate for ethylcellulose or PLGA microcapsules, respectively. Liberated proteins were simultaneously precipitated, pelleted by centrifugation, isolated by decanting the polymer solution, redissolved in 10% w/v sodium dodecyl sulfate in 0.8N sodium hydroxide, and quantified using a modified Lowry assay. Blank microcapsules and exogenously added proteins demonstrated ≥ 93.8% recovery of proteins. The mean encapsulation efficiency of ethylcellulose or PLGA microcapsules was 52.4 or 76.9% for Hb and 86.4 or 74.7% for BSA, respectively. This demonstrates the effective use of centrifugation and the importance of an appropriate cosolvent system in the measure of encapsulation efficiency where one solvent dissolves the polymer while the other solvent quantitatively precipitates the liberated protein. It is evident that an alkaline solution of sodium dodecyl sulfate is efficient at quantitatively dissolving precipitated proteins. Remediation of problems observed with current methods and high reproducibility suggest that this modified method is generally applicable to the measure of protein encapsulation efficiency of polymer microcapsules.

DEVELOPMENT OF A CENTRIFUGATION-BASED PLANT DESIGN TO IMPROVE THE TECHNICAL AND ECONOMIC INDICATORS OF FRITS

The technical solution aimed at reducing the energy loss of the enterprise during drying of the frit is considered: the exclusion of the electric heaters and fan, the use of centrifugation for the developed design of the installation. Analysis of the static and dynamic characteristics of the installation was performed in APM FEM with a uniform load distribution along the bottom of the rotor. The functional problem of smooth starting and braking of the centrifuge drum was solved using an adjustable electric drive with a frequency converter, which provides step control up to 5 rpm. The annual economic effect of the implementation of the installation for drying frits is calculated.

EFFECTIVENESS OF A SIMPLE AND INEXPENSIVE SPERM PROCESSING TECHNIQUE ON INTRAUTERINE INSEMINATION (IUI) PREGNANCY OUTCOMES

Intrauterine insemination (IUI) has been used for the treatment of infertility often as a first-line therapy. Its overall success varies with pregnancy rates/cycle ranging from 5-20%. Sperm processing is required prior to an IUI. Sperm preparation techniques such as density gradient separation (DG) and simple washing (SW) require the use of centrifugation to separate the sperm from the seminal plasma. DG yields a high concentration of motile sperm, relatively free of cellular debris and leukocyte contamination. However, this method is expensive and time-consuming with a low motile sperm recovery rate (20-70% of initial parameters). SW is a fast and inexpensive method of sperm separation using only a single centrifugation wash step. This technique concentrates all cells found in the original sample such as motile sperm, immotile sperm, cellular debris, and leukocytes. Despite the known differences in recovery rates and the ability of separating motile sperm from seminal contaminants, there is limited knowledge about the effect of the different sperm preparation methods on clinical outcomes. To evaluate the efficacy of DG and SW sperm preparation techniques on IUI clinical outcomes. Data on 600 subfertile couples undergoing IUIs at an academic center from 3/2017 to 4/2018 were retrospectively analyzed. Sperm was prepared with DG for 300 specimens and with SW for another 300 specimens. DG Method: Liquified semen was layered over 50:90% ISolate (Fujifilm) and centrifuged at 300g for 20 min. DG sperm pellet was resuspended in 5 ml Multipurpose Handling Medium (MHM; FujiFilm) and centrifuge for 10 min at 300g. This final step was repeated, and the final pellet was resuspended with MHM to a final insemination volume of 0.5 ml. SW Method: Liquified semen was added to 10 ml MHM and centrifuged for 10 min at 300g. SW sperm pellet was resuspended with MHM for a final insemination volume of 0.5 ml. There was no difference in interval between semen production and time of insemination between the 2 groups. Outcome measures: pregnancy rate (PR), livebirth rate (LBR), spontaneous abortion rate (SABR) (out of clinical pregnancies), and multiple pregnancy rate (MPR) (out of livebirths). Parametric and non-parametric tests were used as appropriate. Logistic regression was used to calculate odds ratios, controlling for age. P-values<0.05 were considered statistically significant. There were no significant differences in the demographic and semen analysis parameters between the two groups, except for higher sperm motility in the SW group (44.4% vs. 48.9, p:0.005) (Table 1). PR, LBR, SABR, MPR were not significantly different between the 2 methods: PR (14.7% vs. 16.7%, p:0.500), LBR (12.3% vs. 12.3%, p:1.000), SABR (14.0% vs. 19.6%, p:0.478), and MPR (8.1% vs. 13.5%, p:0.711), respectively for DG vs. SW. Odds ratios for the same rates yielded no significant results, after adjusting for age, as follows: [DG vs. SW, OR (95%CI)] PR [0.884 (0.567-1.379), p:0.588], LBR [1.032 (0.632-1.686), p:0.898], SABR [0.667 (0.215-2.066), p:0.482], and MPR [0.521 (0.112-2.430), p:0.407]. Our results demonstrate that DG and SW are equally effective methods for preparing sperm for IUIs.

Use of Centrifugation to Reduce the Interference Rate of an NMR-Based Lipoprotein Assay

Abstract Physicians are increasingly using nuclear magnetic resonance (NMR) analysis of lipoproteins to assess patients’ cardiovascular disease risk. For certain lipid measurements, like the standard lipid panel, which includes cholesterol, HDL-C, LDL-C, and triglycerides, there is debate about the necessity of fasting prior to a blood draw because the results may still be useful in the nonfasting state. Unfortunately, nonfasting specimens are poorly tolerated in NMR spectroscopy due to spectral overlap of chylomicrons and free fatty acids with other lipoprotein particles. However, it is unknown if valid lipoprotein levels can be extracted from these specimens. Approximately 5% of patient samples submitted for analysis have interferences that prevent analytical deconvolution of the NMR spectral data with the Numares Health AXINON LipoFIT assay. Retrospective review of patient samples from our laboratory showed that the interference rate increased with increasing triglyceride concentrations. Therefore, we hypothesized that sample centrifugation would reduce interference by low-density lipids on the NMR measurement. Preanalytical treatment consisted of centrifugation of 1 mL of serum at 13,000 rcf for 20 minutes followed by carefully pipetting 750 to 800 μL of serum from the lower half of the tube for the NMR measurement, thus avoiding the low-density chylomicrons and lipids at the top. To ensure that centrifugation does not disrupt the measured lipoproteins, a control set of 20 deidentified samples with previous NMR results received our preanalytical treatment and were reanalyzed. To test if centrifugation reduced interferences, a test set of 20 deidentified samples without NMR results due to interference were subjected to the preanalytical treatment and reanalyzed. The pre- and postspin results were analyzed, and the pre- and postspin spectra were compared. Our results showed that, for the control set, the average percent differences for all eight parameters determined by NMR (LDL, HDL, small-LDL, large-HDL, and large-VLDL particle numbers, and particle sizes of LDL, HDL, and VLDL) were within our acceptable limits of ≤10%. The pre- and postspin spectra of the controls were nearly identical. Fourteen of the 20 test samples (70%) achieved results after the preanalytical treatment. Visual inspection of test samples suggested that some, but not all, test samples had chylomicrons present due to the appearance of an upper white layer after centrifugation. The spectral comparison of pre- and postspin samples showed slight changes in the lipoprotein region. In conclusion, our study demonstrates that centrifugation is a promising preanalytical treatment to reduce the interference rate for NMR measurements of lipoproteins. Future goals include expanding this study to a larger sample set and determining if we can better identify samples that may benefit from this preanalytical treatment. In addition, the results suggest that there are other sources of interferences to investigate to improve the performance of the LipoFIT assay.

Spray-dried anti-Campylobacter bacteriophage CP30A powder suitable for global distribution without cold chain infrastructure.

Campylobacter jejuni is a leading cause of foodborne illness globally. In this study, a spray drying and packaging process was developed to produce a thermally-stable dry powder containing bacteriophages that retains biological activity against C. jejuni after long distance shipping at ambient temperature. Spray drying using a twin-fluid atomizer resulted in significantly less (p < 0.05) titer reduction than spray drying using a vibrating mesh nebulizer. The use of centrifugation and dilution of filtered bacteriophage lysate in the formulation step resulted in a significantly greater (p < 0.05) proportion of bacteriophages remaining active relative to use of no centrifugation and dilution. The spray-dried bacteriophage powder generated using leucine and trehalose as excipients was flowable, non-cohesive, and exhibited a high manufacturing yield. The powder retained its titer with no significant differences (p > 0.05) in biological activity after storage in suitable packaging for at least 3 weeks at room temperature and after ambient temperature shipping a total distance of approximately 19,800 km, including with a 38 °C temperature excursion. The bacteriophage powder therefore appears suitable for global distribution without the need for cold chain infrastructure.

Rapid sampling during synthesis of lead halide perovskite nanocrystals for spectroscopic measurement

The photophysical properties of lead halide perovskite nanocrystals (NCs) are critical to their potential application in light emitting devices and other optoelectronics, and are typically characterized using optical spectroscopies. Measurements of nuclei and nascent NC photophysics during synthesis provide insight into how the reaction can be changed to control the properties of the resulting NCs. However, these measurements are typically only performed ex situ after growth is halted by centrifuging the reaction mixture for several minutes. Here, a method is reported to rapidly sample the reaction mixture during a solvation-limited synthesis to enable multiple spectroscopic measurements during nucleation and NC growth. Absorbance and fluorescence measurements of a reaction mixture during the formation of methylammonium lead triiodide perovskite NCs are reported. The changing positions of spectral features as a function of reaction time show the expected weakening of exciton confinement during NC growth. The evolving fluorescence spectra demonstrate that the capping and surface passivation of nascent NCs changes during the reaction. The species in the reaction mixture, particularly during the early stages of the synthesis, are shown to be unstable. This indicates that, even for a relatively slow solvation-limited reaction, the photophysics of the reaction mixture can only be accurately captured if spectroscopic measurements are completed within seconds of sampling. The common use of centrifugation to quench NC syntheses prior to spectroscopic measurement biases the NC population towards more stable, well-capped NCs and does not accurately report on the full NC population in a reaction mixture.