Fractionation Protocol Research Articles

An optimized fractionation method reveals insulin-induced membrane surface localization of GLUT1 to increase glycolysis in LβT2 cells

Insulin is an important regulator of whole-body glucose homeostasis. In insulin sensitive tissues such as muscle and adipose, insulin induces the translocation of glucose transporter 4 (GLUT4) to the cell membrane, thereby increasing glucose uptake. However, insulin also signals in tissues that are not generally associated with glucose homeostasis. In the human reproductive endocrine axis, hyperinsulinemia suppresses the secretion of gonadotropins from gonadotrope cells of the anterior pituitary, thereby linking insulin dysregulation to suboptimal reproductive health. In the mouse, gonadotropes express the insulin receptor which has the canonical signaling response of IRS, AKT, and mTOR activation. However, the functional outcomes of insulin action on gonadotropes are unclear. Here, we demonstrate through use of an optimized cell fractionation protocol that insulin stimulation of the LβT2 gonadotropic cell line results in the unexpected translocation of GLUT1 to the plasma membrane. Using our high purity fractionation protocol, we further demonstrate that though Akt signaling in response to insulin is intact, insulin-induced translocation of GLUT1 occurs independently of Akt activation in LβT2 cells.

Periodic membrane fractionation of freshwater organic matter reveals various reactivity patterns during chlorine/chloramine disinfection

Although drinking water disinfection has significantly reduced mortality from waterborne diseases, the formation of potentially harmful disinfection by-products (DBPs) remains a concern today. These DBPs emerge from the reaction of dissolved organic matter (DOM) with chlor(am)ine during disinfection. Identifying these DBP precursors from heterogeneous and complex DOM mixtures is challenging and to address this, a novel membrane fractionation protocol was developed to isolate and identify the organic DBP precursors from fresh water sources. A tight ultrafiltration and nanofiltration membrane were carefully selected to partition DOM into three molecular weight (MW) fractions: high (>20 kDa), medium (0.3–20 kDa) and low (<0.3 kDa) as defined by high performance-size exclusion chromatography- total organic carbon analysis. A mathematical tool was developed to optimize the fractionation protocol. Therefore, ultrafiltration was executed before nanofiltration and the concentration factor was maximized without inducing fouling to obtain the purest fractions. The mathematical tool was also set in place to predict the necessary diafiltration factor for each individual fractionation experiment based on the initial organic matter composition of the surface water allowing the fractionation protocol to be effective at all times. The protocol was applied to surface water samples collected six times across three seasons yielding a fraction enriched in high MW compounds (up to 50 %), a fraction having more than 80 % of medium MW compounds and a fraction only containing low MW compounds. Although the medium MW fraction showed the highest reactivity for the majority of the investigated DBPs, the low MW fraction showed high reactivity for iodinated trihalomethanes during chlorination and for haloacetonitriles during chloramination. The high MW fraction had the lowest reactivity towards DBPs, which can have important implications for a drinking water treatment since this fraction is generally the most effectively removed by e.g. coagulation, while the more important fractions for DBP formation such as the medium and low MW compounds remain in the water until the disinfection step.

Additional confirmation of successful cell suspension fractionation of metastatic axillary node tissue

We present herein an extension to our recently developed and published method termed "Fractionation of Nodal Cell Suspension" (FNCS). The method enables efficient subcellular fractionation into nuclear (N) and cytosolic (C) compartments of extremely fibrous and problematic metastatic axillary lymph node (mALN) tissue, using the entire nodule. For the purpose of the present study, a case of invasive lobular breast cancer (BC) patient with pT2N3aMx status and defined primary tumor markers (ERα 8, PR-B 8, and HER2 score 0) was available. Initially, the mALN tissue of this patient was analyzed by immunohistochemistry (IHC), and a positive correlation of nodal ERα, PR-B and HER2 biomarkers to those of the primary tumor was obtained. Subsequently, the mALN was FNCS fractionated into N and C, and Western blot (WB) analysis demonstrated a single band for ERα, PR-B and nuclear loading control (HDAC1) in nuclear, but not in the cytosolic compartments, confirming the efficiency of our fractionation protocol. At the same time, HER2 bands were not observed in either compartment, in accordance with HER2 negativity determined by IHC in both primary tumor and mALN tissue. In conclusion, by confirming the nuclear expression of ERα and PR-B biomarkers in metastatic loci, we demonstrate the purity of the FNCS-generated compartments - the protocol that offers a reliable tool for further analysis of nuclear versus cytosolic content in downstream analysis of novel biomarkers in the whole mALN of BC patients.

Exploring the Impact of French Raw-Milk Cheeses on Oxidative Process Using Caenorhabditis elegans and Human Leukocyte Models.

Fermented foods, including cheeses, have garnered increased interest in recent years for their potential health benefits. This study explores the biological properties of eight French raw-milk cheeses-goat cheese, Saint-Nectaire, Cantal, Bleu d'Auvergne, Roquefort, Comté, Brie de Meaux, and Epoisses-on oxidative processes using both in vivo (Caenorhabditis elegans) and in vitro (human leukocytes) models. A cheese fractionation protocol was adapted to study four fractions for each cheese: a freeze-dried fraction (FDC) corresponding to whole cheese, an apolar (ApE), and two polar extracts (W40 and W70). We showed that all cheese fractions significantly improved Caenorhabditis elegans (C. elegans) survival rates when exposed to oxidative conditions by up to five times compared to the control, regardless of the fractionation protocol and the cheese type. They were also all able to reduce the in vivo accumulation of reactive oxygen species (ROS) by up to 70% under oxidative conditions, thereby safeguarding C. elegans from oxidative damage. These beneficial effects were explained by a reduction in ROS production up to 50% in vitro in human leukocytes and overexpression of antioxidant factor-encoding genes (daf-16, skn-1, ctl-2, and sod-3) in C. elegans.

Fractionation and characterization of Dunaliella microalgal biomass and extraction of carotenoids

This paper presents findings on the application of two fractionation protocols of Dunaliella microalgal biomass with the aim to separate and recover the lipid, pigment and protein fraction, leaving as “residue” the fraction that contains carbohydrates. The first protocol (LPC) is based on the initial extraction of lipids, followed by extraction of proteins, while in the second protocol (CLP), the pigments are first extracted followed by lipids extraction. With the LPC protocol the recovered lipid fraction was 34 and 32 wt%, while the recovered proteinic fraction was 46 and 50 wt% for the two samples under study, i.e. 1120 (D. granulata) and 1220 (D. minutissima). The CLP protocol led to the initial extraction of the pigment fraction with a percentage of 10–15 wt% while the subsequently recovered lipidic fraction was 16–24 wt%. The initial biomass was characterized for its moisture (<0.7 wt% for both samples), ash (approx. 45 wt% for both samples), organic matter, elemental composition and higher heating value (HHV) which was 23 and 30 MJ/kg (organic matter based-OMB). The composition of the lipid fraction was analyzed via esterification and gas chromatography (GC-FID) showing C16 and C18 as the predominant acids in the samples. The residue/carbohydrate fraction was analyzed via acid hydrolysis and analysis by high pressure liquid chromatography (HPLC) showing about 20 wt% (-OMB) content of carbohydrates. The high ash content of the biomass was found to be co-extracted during the fractionation protocols at a percentage of ≈49 % in the lipidic fraction and 45 % in the proteinic fraction. Furthermore, emphasis was given on pigment extraction and recovery using various solvents and quantification methods. Measurements of β-carotene, carotenoids, and chlorophyll content through UV and HPLC analyses is presented with chlorophyl and carotenoid content being ≈12 wt% and ≈9 wt% for samples 1120 and 1220 (-OMB). Additionally, a comparison between three solvents (THF, dichloromethane, acetone) was performed regarding their ability to extract the pigments from the microalgal biomass showing that acetone is capable of extracting pigments when concentration of the pigments is not high. The comparison of the HPLC with the UV method, suggested that UV spectroscopy is a reliable and rapid alternative for pigments determination, while HPLC enables identification and quantitative determination of specific pigments and chlorophyll species. The study provides insights for different fractionation protocols applied in two not yet fully studied Dunaliella species as well as the chlorophyl and carotenoid content.

Solvent Fractionation of Technical Lignin Materials with Industrially Relevant Solvents

Lignin fractionation aims at dividing the original lignin material into more uniform fractions that have the qualities desired for further processing of the material. Solvent fractionation is a versatile method that produces a soluble and an insoluble fraction with varying compositions, including molecular weight distribution, polydispersity (PDI), and phenolic hydroxyl (OHph) content. These properties are key factors in the further downstream applicability of lignin. Kraft, organosolv, and hydrolysis lignins were fractionated using multiple solvents and their water solutions with a single-stage fractionation protocol. The produced fractions were analyzed for their molecular size distribution and OHph content. Soluble fractions were discovered with desirable qualities, including a narrow size distribution (lowest PDI 1.4) and accumulation of OHph groups (up to 3.05 mmolOH/glignin). Three fractionation solvents, i.e., pure isopropanol, pure ethanol, and 30% γ-valerolactone, were found to produce solvent-specific soluble fractions that had relatively identical characteristic Mw, Mn, and PDI values regardless of the initial lignin material. This is crucially important new data that is beneficial for robust lignin applicability at industrial scale. Other important observations were made regarding water-alcohol solutions, as the increase in initial solid content resulted in changes in the composition of the fractions produced. Further applicability of the obtained fractions is examined in this paper. Patterns as well as differences between the three lignin materials were observed in their dissolution and the resulting compositions of the soluble fractions. This wide comparable dataset of industrially relevant solvents and lignin materials provides significant insight into the possibilities of lignin downstream processing.

Low-Dose Radiation Therapy Impacts Microglial Inflammatory Response without Modulating Amyloid Load in Female TgF344-AD Rats.

Low-dose radiation therapy (LD-RT) has demonstrated in preclinical and clinical studies interesting properties in the perspective of targeting Alzheimer's disease (AD), including anti-amyloid and anti-inflammatory effects. Nevertheless, studies were highly heterogenous with respect to total doses, fractionation protocols, sex, age at the time of treatment and delay post treatment. Recently, we demonstrated that LD-RT reduced amyloid peptides and inflammatory markers in 9-month-old TgF344-AD (TgAD) males. As multiple studies demonstrated a sex effect in AD, we wanted to validate that LD-RT benefits are also observed in TgAD females analyzed at the same age. Females were bilaterally treated with 2 Gy×5 daily fractions, 2 Gy×5 weekly fractions, or 10 fractions of 1 Gy delivered twice a week. The effect of each treatment on amyloid load and inflammation was evaluated using immunohistology and biochemistry. A daily treatment did not affect amyloid and reduced only microglial-mediated inflammation markers, the opposite of the results obtained in our previous male study. Moreover, altered fractionations (2 Gy×5 weekly fractions or 10 fractions of 1 Gy delivered twice a week) did not influence the amyloid load or neuroinflammatory response in females. A daily treatment consequently appears to be the most efficient for AD. This study also shows that the anti-amyloid and anti-inflammatory response to LD-RT are, at least partly, two distinct mechanisms. It also emphasizes the necessity to assess the sex impact when evaluating responses in ongoing pilot clinical trials testing LD-RT against AD.

Extraction and purification of seaweed protein from Ulva sp. - Challenges to overcome

Ulva sp. has gained attention as a promising and sustainable protein source, due to its high essential amino acid content. This study investigated traditional extraction methods using a double screw press followed by acid or heat precipitation. Furthermore, the screw pressing was combined by a novel purification method that combines mild heat treatment with microfiltration for removal of chlorophyll, to produce white Ulva protein. The double screw press followed by acid precipitation or heat denaturation showed low total protein yields, with 14% of the biomass protein extracted, of which half was soluble. The highest total protein yield (5.5%) was achieved with acid precipitation at pH 2, while heat denaturation at 90 °C resulted in a total protein yield of 4.1%. Acid precipitation proved to be effective with ∼ 81% precipitation of solubilized protein compared to ∼ 43% for heat denaturation. While microfiltration effectively retained chlorophyll in the retentate, the recovery of protein in the permeate remained challenging. SDS-PAGE analysis of proteins in different microfiltration-fractions showed that larger protein complexes (>250 kDa) were unable to transmit through the membranes, resulting in a maximum recovery of 24.9% of the feed-protein in the permeate. Further refinement of Ulva fractionation protocols is imperative for improvement.

Bioassay-Guided Fractionation of Pittosporum angustifolium and Terminalia ferdinandiana with Liquid Chromatography Mass Spectroscopy and Gas Chromatography Mass Spectroscopy Exploratory Study

Bioprospecting native Australian plants offers the potential discovery of latent and novel bioactive compounds. The promising cytotoxic and antibacterial activity of methanolic extracts of Pittosporum angustifolium and Terminalia ferdinandiana led to further fractionation and isolation using our laboratory’s bioassay-guided fractionation protocol. Hence, the aim of this study was to further evaluate the bioactivity of the fractions and subfractions and characterize bioactive compounds using liquid chromatography mass spectroscopy (LC-MS/MS) and gas chromatography MS (GC-MS). Compounds tentatively identified in P. angustifolium Fraction 1 using LC-ESI-QTOF-MS/MS were chlorogenic acid and/or neochlorogenic acid, bergapten, berberine, 8′-epitanegool and rosmarinic acid. GC-MS analysis data showed the presence of around 100 compounds, mainly comprising carboxylic acids, sugars, sugar alcohols, amino acids and monoalkylglycerols. Furthermore, the fractions obtained from T. ferdinandiana flesh extracts showed no cytotoxicity, except against HT29 cell lines, and only Fraction 2 exhibited some antibacterial activity. The reduced bioactivity observed in the T. ferdinandiana fractions could be attributed to the potential loss of synergy as compounds become separated within the fractions. As a result, the further fractionation and separation of compounds in these samples was not pursued. However, additional dose-dependent studies are warranted to validate the bioactivity of T. ferdinandiana flesh fractions, particularly since this is an understudied species. Moreover, LC-MS/GC-MS studies confirm the presence of bioactive compounds in P. angustifolium Fraction 1/subfractions, which helps to explain the significant acute anticancer activity of this plant. The screening process designed in this study has the potential to pave the way for developing scientifically validated phytochemical/bioactivity information on ethnomedicinal plants, thereby facilitating further bioprospecting efforts and supporting the discovery of novel drugs in modern medicine.

Determination of 90Sr in highly radioactive aqueous samples via conversion to a kinetically stable 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex followed by concentration–separation–fractionation based on capillary electrophoresis–liquid scintillation

BackgroundThe Fukushima Daiichi Nuclear Power Plant accident (2011) released large amounts of radioactive substances into the environment and generated highly radioactive debris. Post-accident countermeasures are currently in the phase of fuel debris removal, which requires the analysis of radioactive contaminants in the environment and fuel. The spectra of solely β-emitting nuclides, such as 90Sr, overlap; thus, an effective method for nuclide separation is desired. Since conventional methods for high-dose sample analysis pose substantial exposure risks and generate large amounts of secondary radioactive waste, faster procedures allowing for decreased radiation emission are highly desirable. ResultsIn this study, we developed a 90Sr2+ quantitation technique based on liquid scintillation counting (LSC)–coupled capillary transient isotachophoresis (ctITP), along with two-point detection and relying on the rapid concentration, separation, and fractionation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-complexed 90Sr2+ in a single run. The applicability of our method for the analysis of real-world samples was verified by conducting addition-recovery experiments using a seawater reference material and radioactive liquid waste obtained from the radioactive waste treatment facility at the Japan Atomic Energy Agency. The recovery determined by LSC was 95–113%, indicating successful quantitative analysis. 90Sr recovery was determined to be 90.1% from a contaminated water sample obtained from the Fukushima Daiichi Nuclear Power Plant, which was analyzed using the standard addition of 90Sr. The sensitivity (detection limit = 0.016 Bq) of the proposed method on a radioactivity basis was equal to or higher than that of the conventional method using ion exchange-LSC (0.012–0.07 Bq). Significance and noveltyOur method allows for the handling of high-dose radioactive samples at the microliter level and is substantially faster than conventional ion exchange protocols, whereas ctITP has not been used for practical applications due to inaccurate collection and lack of a suitable chemical system. The concentration–separation–fractionation protocol in ctITP is successful due to the existence of a rare inert Sr2+ complex and precise fractionation. This study establishes a pathway toward safer and more practical analysis of radionuclides.

Predicting Radiotherapy Patient Outcomes with Real-Time Clinical Data Using Mathematical Modelling

Longitudinal tumour volume data from head-and-neck cancer patients show that tumours of comparable pre-treatment size and stage may respond very differently to the same radiotherapy fractionation protocol. Mathematical models are often proposed to predict treatment outcome in this context, and have the potential to guide clinical decision-making and inform personalised fractionation protocols. Hindering effective use of models in this context is the sparsity of clinical measurements juxtaposed with the model complexity required to produce the full range of possible patient responses. In this work, we present a compartment model of tumour volume and tumour composition, which, despite relative simplicity, is capable of producing a wide range of patient responses. We then develop novel statistical methodology and leverage a cohort of existing clinical data to produce a predictive model of both tumour volume progression and the associated level of uncertainty that evolves throughout a patient’s course of treatment. To capture inter-patient variability, all model parameters are patient specific, with a bootstrap particle filter-like Bayesian approach developed to model a set of training data as prior knowledge. We validate our approach against a subset of unseen data, and demonstrate both the predictive ability of our trained model and its limitations.

Proliferation Saturation Index to Simulate Adaptive Radiation Fractionation in HPV-Associated Oropharyngeal Cancer

In current clinical practice for fractionated radiotherapy (RT) patients receive a fixed fractionation protocol throughout radiation course. Radiation oncology has not yet moved toward adaptive fractionation to change RT fractionation based on individual patients' response dynamics. We have previously introduced a novel mathematical and imaging biomarker, proliferation saturation index (PSI), to simulate and evaluate tumor volume dynamics during therapy. Robust tumor volume reduction was associated with lower PSI, and PSI has been utilized to select fractionation schedules for the first mathematical model informed clinical trial. Here we simulate the possibility of adapting the selection of RT fractionation on a week-by-week basis by utilizing a dynamic weekly estimation of PSI. We generated a cohort of in silico patients by sampling from distributions of PSI, radiosensitivity, and growth rate parameters. These distributions were by pre-treatment and on-treatment tumor volume dynamics from a prospective clinical trial (NCT03656133), where 49 HPV+ OPC patients were treated with either 2 Gy daily fractions up to a total of 70 Gy if PS<0.75, or hyperfractionation at 1.2 Gy b.i.d. up to a total of 66 Gy for patients with PSI ≥ 0.75. We then simulated standard (2 Gy q.d.) and hyperfractionated (1.2 Gy b.i.d) for 1-8 weeks of RT to build a library of response dynamics for the different parameter combinations and treatment durations. This library was then used to estimate optimal adaptive fractionation schedules for the in silico patients that maximized tumor volume reduction and minimized total RT dose. We found that PSI evolved towards lower values (i.e., higher proliferative fractions) over the fractionation RT treatment course. For patients that began treatment with standard fractionation, no adaption was found to be superior to uniform fractionation. For selected patients who began treatment with hyperfractionation, however, the optimal fractionation schedule included a period of upfront hyperfractionated RT followed by a switch to standard fractionation at different times during their treatment course. The results of this study suggest that in addition to selecting the best initial fractionation schedule, a novel approach to personalize RT for OPC patients may include adapting the fractionation based on early treatment response dynamics. These results motivate validation in prospective clinical trials.

Irradiation Affects Adipose-Derived Stem Cells and Wound Healing Depending on Radiation Dose and Frequency.

Radiation therapies are often associated with permanent devitalization in the surrounding tissue. The authors hypothesized that stem cells are damaged depending on each irradiation dose and frequency of fractionated radiotherapies, which results in impaired tissue function, including wound-healing capacity. Susceptibility of human adipose-derived stem cells (ASCs) to a single irradiation (0 to 10 Gy) was assessed in vitro. In vivo chronic radiation effects were also assessed on mouse dorsal skin ( n = 4 to 5) for 6 months after a total of 40 Gy irradiation (0 Gy as control) using 1 of 3 fractionated protocols (2 Gy daily for 20 days, 10 Gy weekly for 4 weeks, or 10 Gy monthly for 4 months). Oxygen partial pressure, oxygen saturation of hemoglobin, and dorsal skin viscoelasticity were measured periodically, and wound healing and tissue immunohistology were compared at 6 months. A single irradiation of cultured human ASCs resulted in a dose-dependent increase in cell death up to 2 Gy but with no further increases between 2 and 10 Gy. Most of the apoptotic ASCs were in the proliferation phase. Among the 3 in vivo irradiation protocols, the 2 Gy×20 group had the most severe chronic tissue damage (ie, skin dysfunction, subcutaneous atrophy, depletion of CD34 + stem cells) 6 months after the irradiation. Wound healing was also impaired most significantly in the 2 Gy×20 group. These results have important clinical implications for surgeons and radiotherapists in the timing of surgical interventions and the optimization of fractionation protocols. Irradiation damages stem cells depending on the radiation dose and frequency. Using the ultimately optimized protocol, surgeons can minimize the long-term functional deficits of radiated tissue without losing the anticancer efficacy of radiation therapy.

Predicting the Tumour Response to Radiation by Modelling the Five Rs of Radiotherapy Using PET Images

Despite the intensive use of radiotherapy in clinical practice, its effectiveness depends on several factors. Several studies showed that the tumour response to radiation differs from one patient to another. The non-uniform response of the tumour is mainly caused by multiple interactions between the tumour microenvironment and healthy cells. To understand these interactions, five major biologic concepts called the “5 Rs” have emerged. These concepts include reoxygenation, DNA damage repair, cell cycle redistribution, cellular radiosensitivity and cellular repopulation. In this study, we used a multi-scale model, which included the five Rs of radiotherapy, to predict the effects of radiation on tumour growth. In this model, the oxygen level was varied in both time and space. When radiotherapy was given, the sensitivity of cells depending on their location in the cell cycle was taken in account. This model also considered the repair of cells by giving a different probability of survival after radiation for tumour and normal cells. Here, we developed four fractionation protocol schemes. We used simulated and positron emission tomography (PET) imaging with the hypoxia tracer 18F-flortanidazole (18F-HX4) images as input data of our model. In addition, tumour control probability curves were simulated. The result showed the evolution of tumours and normal cells. The increase in the cell number after radiation was seen in both normal and malignant cells, which proves that repopulation was included in this model. The proposed model predicts the tumour response to radiation and forms the basis for a more patient-specific clinical tool where related biological data will be included.

Insight into corrosion inhibition mechanism of carbon steel by an algal extract as an eco‐friendly corrosion inhibitor in 0.5 M H2SO4: Experimental and molecular dynamics study

AbstractTo explore the potential of new ecological corrosion inhibitors, we investigated the mechanism of corrosion inhibition in carbon steel using Halopitys incurvus, an algal extract available in both crude and chromatographic fractions, in a 0.5 M H2SO4 medium. Various methods were employed, including gravimetry, electrochemical impedance spectroscopy, polarization curves, scanning electron microscopy (SEM) techniques, and molecular dynamics. The crude extract exhibited a high corrosion‐inhibiting potential, with a maximum inhibitory efficiency of 85.30% at 600 mg/L. The electrochemical results indicated mixed behavior of the crude extract. Furthermore, we found that the adsorption of the crude extract onto the metal surface followed the Langmuir isotherm pattern. The SEM study confirmed our proposition that extract molecules were adsorbed onto the carbon steel surface. Using the chromatographic fractionation protocol, we were able to distinguish four main fractions, and the most effective fractions reached an inhibitory efficiency value of 96.00% at 100 mg/L. The molecular dynamics simulation confirmed the experimental results.

PO-1213 A comparative analysis of two fractionation protocol of simultaneous integrated boost-IMRT for LAHNC

PO-1213 A comparative analysis of two fractionation protocol of simultaneous integrated boost-IMRT for LAHNC

Specification of hemocyte subpopulations based on immune-related activities and the production of the agglutinin MkC1qDC in the bivalve Modiolus kurilensis

Bivalves, such as Modiolus are used as indicator organisms to monitor the state of the marine environment. Even though hemocytes are known to play a key role in the adaptive and protective mechanisms of bivalves, these cells are poorly studied in horse-mussel Modiolus kurilensis. In this paper, we present classification of horse-mussel hemocytes based on their immune functions, including the production of specific immune-related molecules, as well as their morphological composition after isolation by density gradient centrifugation. An effective fractionation protocol was adapted to separate four hemocyte subpopulations with distinct morphofunctional profiles. First subpopulation consisted of small under-differentiated hemoblasts (2.20 ± 0.85%) with a bromodeoxyuridine positive nucleus, and did not show any immune reactivity. Second was represented by agranulocytes (24.11 ± 2.40%), with evenly filled cytoplasm containing a well-developed protein-synthesizing apparatus, polysomes, smooth endoplasmic reticulum and mitochondria, and positively stained for myeloperoxidase, acidic proteins, glycogen and neutral polysaccharides. Third subpopulation consisted of eosinophilic granulocytes (62.64 ± 9.32%) that contained the largest number of lysosomes, peroxisomes and vesicles with contents of different density, and showed the highest phosphatase, reactive oxygen species (ROS) and phagocytic activities. Lastly, fourth group, basophilic granulocytes (14.21 ± 0.34%), are main producers of lectin-like protein MkC1qDC, recently discovered in M. kurilensis and characterized by pronounced antibacterial and anticancer activity. These cells characterized by intracytoplasmic of the MkC1qDC localization, forming granule-like bodies visualized with specific antibody. Both granulocytes and agranulocytes showed phagocytic activity and ROS production, and these reactions were more pronounced for eosinophilic granulocytes, suggesting that this group is the key element of the cell-mediated immune response of M. kurilensis. Our results support a concept of bivalve's hemocyte specification with distinct phenotypes.

Alterations in the LRRK2-Rab pathway in urinary extracellular vesicles as Parkinson’s disease and pharmacodynamic biomarkers

Expression or phosphorylation levels of leucine-rich repeat kinase 2 (LRRK2) and its Rab substrates have strong potential as disease or pharmacodynamic biomarkers. The main objective of this study is therefore to assess the LRRK2-Rab pathway for use as biomarkers in human, non-human primate (NHP) and rat urine. With urine collected from human subjects and animals, we applied an ultracentrifugation based fractionation protocol to isolate small urinary extracellular vesicles (uEVs). We used western blot with antibodies directed against total and phosphorylated LRRK2, Rab8, and Rab10 to measure these LRRK2 and Rab epitopes in uEVs. We confirm the presence of LRRK2 and Rab8/10 in human and NHP uEVs, including total LRRK2 as well as phospho-LRRK2, phospho-Rab8 and phospho-Rab10. We also confirm LRRK2 and Rab expression in rodent uEVs. We quantified LRRK2 and Rab epitopes in human cohorts and found in a first cohort that pS1292-LRRK2 levels were elevated in individuals carrying the LRRK2 G2019S mutation, without significant differences between healthy and PD groups, whether for LRRK2 G2019S carriers or not. In a second cohort, we found that PD was associated to increased Rab8 levels and decreased pS910-LRRK2 and pS935-LRRK2. In animals, acute treatment with LRRK2 kinase inhibitors led to decreased pT73-Rab10. The identification of changes in Rab8 and LRRK2 phosphorylation at S910 and S935 heterologous phosphosites in uEVs of PD patients and pT73-Rab10 in inhibitor-dosed animals further reinforces the potential of the LRRK2-Rab pathway as a source of PD and pharmacodynamic biomarkers in uEVs.

A Production and Fractionation Protocol for Polyvinyl Chloride Microplastics

Concerns about the presence of microplastics in the environment has increased in recent years, prompting more attention from scientists. Thorough exposure studies using artificially produced microplastics containing additives are required to assess their potentially hazardous effects. Therefore, an efficient microplastic production and fractionation protocol was established using a cryogenic grinding and wet-sieving approach. The developed cryogenic grinding method was able to produce (20-40 g/h) polyvinyl chloride (PVC) microplastics having a volume-weighted mean particle size of 391 µm and a span of 2.12. Performing a second grinding cycle on the same particles resulted in microplastics which were smaller (volume-weighted mean size = 219 μm) and had a narrower particle size distribution (span = 1.59). In addition, the microplastics were also fractionated into different particle size ranges using a vibrating wet-sieving tower. The latter technique allowed separating 10 g of PVC microplastics into seven different fractions using six sieves (Ø 200 mm) for 30 min while shaking. By using the developed method, PVC microplastics could easily be made and fractionated into desired particle-size ranges. The proposed protocol could also be adjusted to produce and fractionate microplastics of other plastics.

Cellular Fractionation to Characterize the Interaction of Nucleolin with Alpha-COP

Background and Hypothesis:The goal was to further characterize the interaction between Nucleolin and the alpha subunit of the COPI coatomer complex. Nucleolin contains a C-terminal dilysine motif, which mediates interactions between the WD40 domain of alpha-COP and COPI-interacting proteins. Previous work in the lab showed that this C-terminal dilysine is required for co-immunoprecipitation of alpha-COP with Nucleolin. Because alpha-COP is exclusively found in the cytoplasmic compartment, we hypothesized that the interaction between alpha-COP and Nucleolin is exclusively cytoplasmic but previous co-immunoprecipitations had only been performed from whole cell lysates. Alpha-COP has been shown to bind mRNA, but it was unclear whether this interaction was direct or whether alpha-COP was binding an RNA binding protein (RBP) which would act as a bridge between the mRNA and alpha-COP. Nucleolin acts as an RBP in both the nuclear and cytoplasmic compartments. We hypothesize that some of the mRNA bound to alpha-COP are present due to their association with Nucleolin.&#x0D; Experimental Design or Project Methods:The first aim of the project was to optimize a reproducible cell fractionation protocol to reliably separate nuclear and cytoplasmic compartments. The second goal of the project was to identify mRNA that would co-immunoprecipitated with Nucleolin in HEK293T cells where we can easily express tagged versions of alpha-COP. Nucleolin-bound mRNA had previously been identified in Hela cells. We began by testing for the expression of these mRNAs in 293-TT cells using the published RT-PCR primers.&#x0D; Results:After these tests identified Ftl (Ferritin light polypeptide) as a highly abundant transcript in 293-TTs, we performed RNA immunoprecipitation from cells expressing epitope tagged Nucleolin or alpha-COP. We confirmed that both Nucleolin and alpha-COP are in complex with Ftl mRNA.&#x0D; Potential Impact:Future experiments will use short-hairpin RNA to knockdown Nucleolin and determine whether the levels of Ftl mRNA that co-immunoprecipitated with alpha-COP are reduced in the absence of Nucleolin.