Ultrafiltration Method Research Articles

Evaluation of clarification process of Indonesia’s sweet sorghum juice by ultrafiltration membrane and the physicochemical and sensorial properties of the sugar product

The juice derived from Sorghum bicolor L. exhibits a notable sugar content, rendering it a promising candidate for use as a sweetening agent. The presence of impurities in sorghum juice encompasses several substances such as non-sugar molecules, non-fermentable reducing agents, starch, and minerals like salt, magnesium, and calcium. The objective of this study was to investigate the impact of ultrafiltration (UF) membranes on the physicochemical properties of sorghum juice and sugar granules, as well as to identify customer preferences for these products. The study consisted of three primary phases. The first phase involved investigating the impact of 0–3 cycles of UF treatment on the physicochemical properties of the sorghum juice. The second stage involved comparing the characteristics of sugar granules obtained through UF and non-ultrafiltration (non-UF) methods. The third one focused on examining the preference of sorghum sugar granules in their natural state and as a sweetener for steeped black tea. A 5% significance level was employed in this work to conduct multivariate methodology of variance and the Friedman nonparametric test. The findings of the study indicate that the use of UF treatment has a simultaneous impact on the turbidity and color of sorghum juice. Non-UF sorghum sugar granules exhibit a superior aroma in comparison to UF granules while maintaining identical color, taste, and texture. Consumers exhibited a preference for steeped black tea sweetened with palm sugar, followed by rock sugar, granulated sugar, UF sorghum sugar granules, and non-UF.

The synergistic enhancement or inhibition of different molecular weight components of mannoproteins after ultrafiltration on the encapsulation.

This study investigated the effects of molecular weight regulation on mannoproteins (MPs) in encapsulating both monomeric and oligomeric proanthocyanidins (MOPC). To achieve this, two different conformations of MPs were fractionated by ultrafiltration into two main molecular weight components. The results indicated that regulating molecular weight through ultrafiltration altered the conformation of MPs chains, which in turn affected the intermolecular forces with MOPC. Specifically, the interactions among the different molecular weight components of MPs formed a cross-linking network. High molecular weight components provided external support to this network, while low molecular weight components contributed to internal stabilization. The dense, flexible, and irregular coil structure of MPs facilitated the entry of MOPC into the internal spaces of the cross-linking network. Additionally, protein unfolding into a more stable β-sheet structure further stabilized MPs' cross-linking network, enhancing its capacity to encapsulate MOPC. The presence of more hydrophobic regions and positive charges within the proteins increased the number of binding sites for MOPC. This study aimed to identify the primary structural forms of MPs with potent initial bitter inhibition capabilities on MOPC, and a proposed ultrafiltration method was introduced to modify the triple helix structure of polysaccharide, offering a foundational theoretical framework for enhancing the functional attributes of current or prospective MPs. Additionally, the mixed regulation of polysaccharides with varying conformations was also beneficial for guiding the production of high-efficiency polysaccharide products.

Plasma protein binding of arsenic species in acute promyelocytic leukemia patients and their relationships with hepatic and renal function

ABSTRACT Objectives Percentage protein binding (%PB) of arsenic species in acute promyelocytic leukemia (APL) patients treated with arsenic trioxide remains unclear. It can be different depending on the status of hepatic or renal function. Methods This study obtained steady-state blood samples from normal APL patients and those with hepatic or renal impairment. %PB of inorganic arsenic (iAs), monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV) was determined by analyzing free and total plasma concentrations using ultrafiltration method by HPLC-HG-AFS. Results There is a linear relationship between free and total plasma concentrations for iAs (r2 = 0.952), MMAV (r2 = 0.603), and DMAV (r2 = 0.945). For patients with normal hepatic and renal function, mean %PB was as follows: iAs at 26.7 ± 14.3%, MMAV at 53.3 ± 11.9%, and DMAV at 24.7 ± 7.8%. %PB decreased in patients with renal impairment, with MMAV and DMAV showing statistically significant differences (p < 0.05 for MMAV, p < 0.01 for DMAV). No significant differences in %PB between normal and hepatic impairment group were observed. Conclusion Free arsenic species fraction can be estimated by total concentration. DMAV and iAs present low %PB, while MMAV exhibits a relatively high %PB in plasma. Level of %PB is more likely to be affected by renal function and age.

Influence of boric acid on the speciation of iron (III) in aqueous solutions

During the work, the speciation of iron (III) was studied at the concentration of ~ 1∙10–5 mol/dm3 in aqueous solutions in the presence of boric acid at a concentration of 5–20 g/dm3 in a wide pH range using ion exchange, ultrafiltration and centrifugation methods. The regions of existence of ionic and nonionic forms of iron (III) were determined by ion exchange in solutions with different boric acid concentrations. Experimental data have shown that the pH of the onset of iron (III) colloid formation shifts in the presence of boric acid. The formation of complex compounds of iron (III) with polyborate ions in the neutral – slightly alkaline pH region was established by ultrafiltration and centrifugation. As shown in the paper, the increase in the concentration of boric acid in solution leads to the increase in the region of existence of the supposed complexes. The identified features of the behavior of iron (III) in solutions containing boric acid determine the possibility of using ion exchange and membrane separation methods for purifying solutions. Information about the physical-chemical properties of various metal-ions can provide optimal conditions for the purification of liquid radioactive waste at various nuclear industry facilities.

Combining virus-based affinity ultrafiltration method with serum pharmacochemistry to identify the antiviral pharmacodynamic substances in licorice

Ethnopharmacological relevanceLiorice (Glycyrrhiza uralensis Fisch.), a widely used Chinese herbal medicine, is frequently employed in clinical practice to treat viral pneumonia. However, the pharmacodynamic substances and mechanisms of action responsible for its antiviral effects against H1N1 and RSV remain unclear. Aim of the studyTo investigate the antiviral effects of licorice against H1N1 and RSV. Building on this, we aimed to more comprehensively and accurately identify the pharmacodynamic substances in licorice responsible for its antiviral activity and mechanisms of action against these two viruses. Materials and methodsFirstly, the antiviral effects of licorice against H1N1 and RSV were confirmed through in vivo and in vitro experiments. Then, a combination of virus-based affinity ultrafiltration method (VAUM) and serum pharmacochemistry were used to screen for pharmacological substances in licorice and identify their molecular targets against H1N1 and RSV. Results: The in vivo experiments showed that licorice effectively alleviates H1N1 and RSV induced weight loss and lung tissue damage in mice, while also reducing viral loads of H1N1 and RSV in the lungs. Subsequent in vitro experiments confirmed the presence of original compounds in licorice that directly inhibit H1N1 and RSV. By combining both methods, glycyrrhizic acid, glycyrrhetinic acid (GA), isoliquiritigenin (ISL), and glyasperin A (targeting the M2 ion channel) were ultimately identified as the pharmacodynamic substances in licorice responsible for anti-H1N1 activity. Additionally, licochalcone A (LCA) and glyasperin A, which target RSV surface proteins, were identified as the pharmacodynamic substances responsible for anti-RSV activity. ConclusionsTraditional Chinese medicine (TCM) exerts its antiviral effects through a 'multi-component, multi-target' mechanism, which poses challenges for single active compound screening methods to adequately address. By integrating VAUM and serum pharmacochemistry for the first time, one approach focused on identifying compounds in TCM that directly bind to viral surface proteins, while the other targeted compounds that enter the bloodstream in their original form and exhibit antiviral activity. This provides a novel approach for studying the pharmacodynamic substances of antiviral effects in TCM.

Очистка торфяных грунтовых вод методом ультрафильтрации и ионного обмена

Purpose: assessing the effectiveness of peat groundwater purification using a combination of two methods of ultrafiltration and membrane purification. Identify specific characteristics of the composition of peat or groundwater, determine possible possible methods of treating these waters to ensure compliance with the requirements of SanPiN 2.1.3684–21 “Sanitary and epidemiological requirements for the maintenance of territories of urban and rural settlements, for water bodies, drinking water and drinking water supply to the population, atmospheric air, soil, residential premises, operation of industrial and public premises, organization and implementation of sanitary and anti-epidemic (preventive) measures” to the quality of drinking water. Determine the list of composition indicators that require cleaning. Conduct an assessment of wastewater treatment methods used to treat peat groundwater. Togo, Conduct research on the composition of purified groundwater before and after treatment, determine the effectiveness of the treatment methods used according to various indicators. Determination of the concentrations of groundwater composition components was carried out using approved methods in accordance with GOST R 57164-2010 clause 5; GOST R 57164-2010 clause 6; PND F 14.1:2:4.207-04; PND F 14.1.2:3:4.121-97; PND F 14.1:2:4.114-97; GOST 31594-2012 Method A; PND F 14.1:2:4.50-96; PND F 14.1.2:3:4.111-97; PND F 14.1.2:3:4.111- 97; PND F 14.1.2:3.154-99; PND F 14.1.2:3:4.123-97; GOST 31859-2012; PND F 14.1.2:3:4.4-95; GOST 31940-2012 clause 6; PND F 14.1.2:4.178-02; PND F 14.1.2:4.214-06. An analysis of the results of laboratory studies of the composition of peat groundwater before and after treatment was carried out, and the effectiveness of ultrafiltration and membrane water purification methods was determined. The results of studies of the effectiveness of purification of peat groundwater using ultrafiltration and membrane purification methods show that purified groundwater meets the sanitary and epidemiological requirements for the quality of drinking and domestic water supply established by SanPiN 2.1.3684-21. Practical significance: the results obtained from studies of the effectiveness of peat groundwater treatment using ultrafiltration and membrane treatment methods indicate the possibility of using these methods, because they ensure that water quality meets the requirements of SanPiN 2.1.3684-21.

Natural organic matter (NOM) can increase the uptake fluxes of three critical metals (Ga, La, Pt) in a unicellular green alga

Critical metals such as gallium, lanthanum and platinum are considered essential in a modern economy and for the required energy transition. Their relatively recent and increasing use in new technologies have led to an increase in their environmental mobility. As they reach aquatic systems, these metals can interact with organic ligands and especially Natural Organic Matter (NOM). The formation of organic complexes would be expected to reduce metal bioavailability and uptake by living cells, according to the Biotic Ligand Model (BLM). However, exceptions to this model have been determined for several critical metals in the past. The present work compared internalization kinetics of Ga, La and Pt in the green alga Chlamydomonas reinhardtii in the presence of NOMs from different origins: humic and fulvic acids from Suwannee River as well as NOMs from Ontario (Bannister Lake and Luther Marsh). Complexation was determined using a partial ultrafiltration method allowing for a normalization of data based on speciation to compare all conditions based on the concentration of the metal that was not bound to NOM. While internalization metal fluxes varied greatly from one NOM source to the other, uptake was almost always significantly higher than expected based on metal speciation. Quite often, metal internalization fluxes were even significantly increased in the presence of NOM, for the same total metal exposure concentration. For instance, Pt internalization was twice greater in the presence of Bannister Lake NOM than it was in the absence of NOM. The assumption that such exceptions could be explained by NOM characteristics was contradicted by the variable results from one metal to another. To further explore this phenomenon, internalization mechanisms for these individual metals need to be elucidated. This is a necessary step to accurately estimate the risk posed by the presence of these metals in humic aquatic systems.

Novel Diclofenac-NO Donor With High Affinity for Human Serum Albumin Induces Endoplasmic Reticulum Stress-mediated Cell Death in Human Pancreatic Cancer Cells.

Nitric oxide (NO) has various physiological activities. In this study, diclofenac (DF) which has a high affinity for human serum albumin (HSA) was nitrosylated to a novel NO donor (NDF). The cytotoxic effects and the mechanism of NDF were investigated. Binding experiments of NDF to HSA were performed by the ultrafiltration method. NO was measured by the Griess method. The number of dead cells were measured using annexin V. Apoptosis and endoplasmic reticulum stress were evaluated by western blotting. NDF competitively inhibits the binding of DF to HSA, suggesting that NDF and DF have equivalent binding characteristics. NDF rapidly released NOx after being dissolved. At 200 μM, NDF induced cell death in human pancreatic cancer cells. Western blotting showed that NDF promoted the cleavage of PARP, caspase-3, and caspase-7. Inhibitors of caspase-1 and caspase-9 significantly suppressed NDF-induced cell death, as did a non-specific caspase inhibitor (Z-VAD). In addition, NDF significantly increased the expression of the endoplasmic reticulum stress marker, CHOP. NDF induces apoptotic cell death by causing endoplasmic reticulum stress. The findings of this study suggest that NDF may become a promising compound for the treatment of pancreatic cancer.

Definition of a concentration and RNA extraction protocol for optimal whole genome sequencing of SARS-CoV-2 in wastewater (ANRS0160)

Monitoring the presence of RNA from emerging pathogenic viruses, such as SARS-CoV-2, in wastewater (WW) samples requires suitable methods to ensure an effective response. Genome sequencing of WW is one of the crucial methods, but it requires high-quality RNA in sufficient quantities, especially for monitoring emerging variants. Consequently, methods for viral concentration and RNA extraction from WW samples have to be optimized before sequencing. The purpose of this study was to achieve high coverage (≥ 90 %) and sequencing depth (at least ≥200×) even for low initial RNA concentrations (< 105 genome copies (GC)/L) in WW. A further objective was to determine the range of SARS-CoV-2 RNA concentrations that allow high-quality sequencing, and the optimal sample volume for analysis. Ultrafiltration (UF) methods were used to concentrate viral particles from large influent samples (up to 500 mL). An RNA extraction protocol using silica beads, neutral phenol-chloroform treatment, and a PCR inhibitor removal kit was chosen for its effectiveness in extracting RNA and eliminating PCR inhibitors, as well as its adaptability for use with large influent samples. Recovery rates ranged from 24 % to 63 % (N = 17) for SARS-CoV-2 naturally present in WW samples. 200 mL WW samples can be enough for UF concentration, as they showed high quality sequencing analyses with between 5 × 104 GC/L and 6 × 103 GC/L. Below 6 × 103 GC/L, high-quality sequencing was also achieved for ∼40 % of the samples using 500 mL of WW. Sequencing analysis for variant detection was performed on 200 mL WW samples with coverage of >95 % and sequencing depth of >1000×. Analyses revealed the predominance of variant EG.5, known as Eris (66 %–100 %). The use of UF methods in combination with a suitable RNA extraction protocol appear promising for sequencing enveloped viruses in WW in a context of viral emergence.

Evaluating approach uncertainties of quantitative detection of SARS-CoV-2 in wastewater: Concentration, extraction and amplification

Substantial uncertainties pose challenges to the accuracy of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quantification in wastewater. We conducted a comprehensive evaluation of two concentration methods, three nucleic acid extraction methods, and the amplification performance of eight primer-probe sets. Our results showed that the two concentration methods exhibited similar recovery rates. Specifically, using a 30 kDa cut-off ultrafilter and a centrifugal force of 2500 g achieved the highest virus recovery rates (27.32 ± 8.06 % and 26.37 ± 7.77 %, respectively), with lower corresponding quantification uncertainties of 29.51 % and 29.47 % in ultrafiltration methods. Similarly, a 15 % PEG concentration with 1.5 M NaCl markedly improved virus recovery (26.76 ± 5.92 % and 28.47 ± 6.74 %, respectively), and reducing variation to 22.16 % and 23.66 % in the PEG precipitation method. Additionally, employing a vigorous bead-beating approach at 6 m/s during viral RNA extraction significantly increased RNA yield, with an efficiency reaching up to 82.18 %. Among the evaluated eight primer-probe sets, the E_Sarbeco primer-probe set provided the most stable and consistent quantitative results across various sample matrices. These findings are crucial for establishing robust viral quantification protocols and enhancing methodological precision for effective wastewater surveillance, enabling sensitive and precise detection of SARS-CoV-2.

Improving the technology of “Dutch” cheese through the use of retentate obtained by ultrafiltration method

Improving the technology of “Dutch” cheese through the use of retentate obtained by ultrafiltration method

Bioaffinity Ultrafiltration Combined with HPLC-ESI-qTOF-MS/MS for Screening Potential Bioactive Components from the Stems of Dendrobium fimbriatum and In Silico Analysis.

Dendrobium fimbriatum is a perennial herb, and its stems are high-grade tea and nourishing medicinal materials. Various solvent extracts of D. fimbriatum were evaluated for their anti-inflammatory, anti-acetylcholinesterase (AChE), antioxidant, and anti-α-glucosidase properties. Acetone and EtOAc extracts showed significant antioxidant effects. Acetone, n-hexane, and EtOAc extracts revealed potent inhibition against α-glucosidase. EtOAc, n-hexane, and dichloromethane extracts displayed significant anti-AChE activity. Among the isolated constituents, gigantol, moscatin, and dendrophenol showed potent antioxidant activities in FRAP, DPPH, and ABTS radical scavenging tests. Moscatin (IC50 = 161.86 ± 16.45 μM) and dendrophenol (IC50 = 165.19 ± 13.25 μM) displayed more potent anti-AChE activity than chlorogenic acid (IC50 = 236.24 ± 15.85 μM, positive control). Dendrophenol (IC50 = 14.31 ± 3.17 μM) revealed more efficient anti-NO activity than quercetin (positive control, IC50 = 23.09 ± 1.43 μM). Analysis of AChE and iNOS inhibitory components was performed using molecular docking and/or the bioaffinity ultrafiltration method. In bioaffinity ultrafiltration, the binding affinity of compounds to the enzyme (acetylcholinesterase and inducible nitric oxide synthase) was determined using the enrichment factor (EF). Among the main components of the EtOAc extract from D. fimbriatum stem, moscatin, dendrophenol, gigantol, and batatasin III with acetylcholinesterase exhibited the highest binding affinities, with affinity values of 66.31%, 59.48%, 54.60%, and 31.87%, respectively. Moreover, the affinity capacity of the identified compounds with inducible nitric oxide synthase can be ranked as moscatin (88.99%) > dendrophenol (65.11%) > gigantol (44.84%) > batatasin III (27.18%). This research suggests that the bioactive extracts and components of D. fimbriatum stem could be studied further as hopeful candidates for the prevention or treatment of hyperglycemia, oxidative stress-related diseases, and nervous disorders.

Review of the concentration procedures used to determine the parasitological water quality in Latin America (2000–2022)

ABSTRACT The aim was to review the methodologies used in the concentration of intestinal parasites in different water samples in Latin America. The period of the review was between September and November 2022. Of the total number of articles (N = 87), the search engine with the most articles matching the specified keywords was Google Scholar (56.3% = 49 articles), and Brazil was the country that published the most articles (28.7%). Most of the studies were conducted for surface waters (75.9%) and used methodologies with physical separation without subsequent parasitological concentration (76.9%). However, the ultrafiltration method (UFM) with subsequent parasitological concentration recovered a greater number and type of parasite species. The descriptive study of the review shows that the procedure of using UFM with a parasitological method of flotation and sedimentation would allow an efficient concentration of the parasite species present in the water sample.

Modified Ultrafiltration Technique in Valvular Heart Surgery: A Case Series

Abstract Application of cardiopulmonary bypass (CPB) in cardiac surgery may cause systemic inflammatory reactions, possibly causing clinical sequelae and multiple organ failures such as systolic or diastolic dysfunction, regional or global left ventricle or right ventricle low contractility, and the function of vascular tone. The use of drugs (i.e., corticosteroids) and the ultrafiltration method during the CPB application has been known to prevent or minimize the complication of CPB in cardiac surgery. One of the non-pharmacological approaches to minimize those inflammatory responses is the modified ultrafiltration (MUF) technique. Theoretically, this is due to ultrafiltration which may reduce the interstitial fluid and inflammatory mediator. MUF can be used to counteract inflammatory responses in pediatric cardiac surgery. This ultrafiltration method became a recommended procedure in minimally invasive surgery and conventional open-heart surgery. However, studies about effectiveness in adults are limited. In this case series, the MUF technique was used in three patients who underwent valvular heart surgery with a CPB machine. Following the application of the CPB, the MUF technique is performed for 10 min at a speed of no more than 10% full flow. Hemodynamic measurements and interleukin-6 (IL-6) levels were taken during surgery and 24 h post-CPB. At 24 h post-CPB, the vasoactive inotropic score and duration of mechanical support use were less than 20 and 18–24 h, respectively, and the IL-6 level ranged from 7.22 to 14.22 pg/mL. The cardiac index increased, ranging from 2.43 to 3.47 L/min/m2, and pulmonary vascular resistance decreased, ranging from 78 to 146 dyne s/cm5.

Identification and Molecular Mechanism of Anti-inflammatory Peptides Isolated from Jack Bean Protein Hydrolysates: in vitro Studies with Human Intestinal Caco-2BBe Cells

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.

Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phage-scFv cocktail/ kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Impact of homocysteine on acute ischemic stroke severity: possible role of aminothiols redox status

BackgroundAcute ischemic stroke (AIS) is one of the most common cerebrovascular diseases which accompanied by a disruption of aminothiols homeostasis. To explore the relationship of aminothiols with neurologic impairment severity, we investigated four aminothiols, homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CG) and glutathione (GSH) in plasma and its influence on ischemic stroke severity in AIS patients.MethodsA total of 150 clinical samples from AIS patients were selected for our study. The concentrations of free reduced Hcy (Hcy), own oxidized Hcy (HHcy), free reduced Cys (Cys), own oxidized Cys (cysteine, Cyss), free reduced CG (CG) and free reduced GSH (GSH) were measured by our previously developed hollow fiber centrifugal ultrafiltration (HFCF-UF) method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The concentration ratio of Hcy to HHcy (Hcy/HHcy), Cys to Cyss (Cys/Cyss) were also calculated. The neurologic impairment severity of AIS was evaluated using National Institutes of Health Stroke Scale (NIHSS). The Spearman correlation coefficient and logistic regression analysis was used to estimate and perform the correlation between Hcy, HHcy, Cys, Cyss, CG, GSH, Hcy/HHcy, Cys/Cyss and total Hcy with NIHSS score.ResultsThe reduced Hcy and Hcy/HHcy was both negatively correlated with NIHSS score in AIS patients with P = 0.008, r=-0.215 and P = 0.002, r=-0.249, respectively. There was no significant correlation of Cys, CG, GSH, HHcy, Cyss, Cys/Cyss and total Hcy with NIHSS score in AIS patients with P value > 0.05.ConclusionsThe reduced Hcy and Hcy/HHcy, not total Hcy concentration should be used to evaluate neurologic impairment severity of AIS patient.

#2722 Hemodialysis vascular access flow assessment by ultrasound dilution method (NephroFlow™) and ultrafiltration method with NIKKISO DBB-EXA™

Abstract Background and Aims A functioning vascular access (VA) is needed to ensure optimal dialysis. Reduced or falling levels of VA flow volume (Qa) indicate VA dysfunction and predict thrombosis. Qa can be measured by a number of different techniques, most of all using dilution techniques such as ultrasound dilution to measure the amount of recirculation induced by reversal of blood lines. The NephroFlow™ hemodialysis monitor estimates Qa using ultrasound velocity measurements of flowing blood and their dilution with 0.9% NaCl. Nikisso DBB-EXATM dialysis machine has the capacity to measure the VA recirculation rate by photometry performing a controlled rise on ultrafiltration rate; Qa is subsequently estimated using the Krivitski formula. There is little literature on the comparison of the different methodologies, but it was described that the DBB-EXATM method underestimates Qa compared to others techniques [1]. Our study aims to compare the Qa between the NephroFlow™ ultrasound dilution technique and the Nikisso DBB-EXATM ultrafiltration method. Method We performed an observational study that included 45 hemodialysis patients followed at our unit. Measurements of VA flow using the NephroFlow® device and the Nikisso-EXATM Ultrafiltration method were performed in patients with a native arteriovenous fistula or a graft. They were hemodynamically stable without acute clinical conditions. Both studies were undertaken during the first 90 minutes of a mid-week dialysis session, under a constant dialysis blood pump flow with the ultrafiltration interrupted during the procedure after having reversed the dialysis lines. For each patient we calculated the average of two Qa evaluations conducted at Nikisso-EXATM, spaced 15 days apart, with an additional evaluation using NephroFlow® conducted between these assessments. Results Forty-five patients (male: 71.1%) with a mean age of 73.2 ± 8 years were selected. The main CKD causes were diabetic kidney disease (n = 13, 28.9%) and uncertain etiology (n = 13, 28.9%). The dialysis vintage of these patients was 142.6 months (minimum 19, maximum 168). An arteriovenous fistula was present in 38 patients (84.4%) and an arteriovenous graft in 7 patients (15.6%). In the entire group, the mean flow with NephroFlow™ was 986.9 ± 515 ml/min and with DBB-EXATM 859.7 ± 522 ml/min. The measured values of the two methods presented a significant positive correlation (by Pearson) (R = 0.822, P &amp;lt; 0.01) (Fig. 1). In the group of patients with arteriovenous fistula (n = 38, 84.4%), the mean flow with NephroFlow™ was 961.8 ± 395 ml/min and with DBB-EXATM 839.1 ± 577 ml/min. The measured values of the two methods presented a significant positive correlation (by Pearson) (R = 0.844, P &amp;lt; 0.01). In the group of patients with arteriovenous graft (n = 7, 15.6%), the mean flow with NephroFlow™ was 1017.3 ± 401 ml/min and with DBB-EXATM 909.2 ± 604 ml/min. The measured values of the two methods presented a significant positive correlation (by Pearson) (R = 0.906, P &amp;lt; 0.01). Conclusion We found a significantly positive correlation between the Qa measurements obtained using both methods. The measurement of Qa using the ultrafiltration method on Nikisso DBB-EXATM seems to be a valuable and easily implementable tool for VA surveillance in both arteriovenous fistula and grafts. However, a bigger number of studies are required in this field in order to clarify this correlation with an additional comparative analysis to assess the reproducibility of measurements.

Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries

Background and aimsOvum pick-up (OPU) is an intrinsic step of in vitro fertilization procedures. Nevertheless, it can cause ovarian lesions and compromise female fertility in bovines. Recently, we have shown that intraovarian injection of adipose-derived mesenchymal stromal cells (AD-MSCs) effectively preserves ovarian function in bovines. Given that MSC-derived extracellular vesicles (MSC-EVs) have been shown to recapitulate several therapeutic effects attributed to AD-MSCs and that they present logistic and regulatory advantages compared to AD-MSCs, we tested whether MSC-EVs would also be useful to treat OPU-induced lesions. MethodsMSC-EVs were isolated from the secretome of bovine AD-MSCs, using ultrafiltration (UF) and ultracentrifugation methods. The MSC-EVs were characterized according to concentration and mean particle size, morphology, protein concentration and EV markers, miRNA, mRNA, long noncoding RNA profile, total RNA yield and potential for induction of the proliferation and migration of bovine ovarian stromal cells. We then investigated whether intraovarian injection of MSC-EVs obtained by UF would reduce the negative effects of acute OPU-induced ovarian lesions in bovines. To do so, 20 animals were divided into 4 experimental groups (n = 5), submitted to 4 OPU cycles and different experimental treatments including vehicle only (G1), MSC-EVs produced by 7.5 × 106 AD-MSCs (G2), MSC-EVs produced by 2.5 × 106 AD-MSCs (G3) or 3 doses of MSC-EVs produced by 2.5 × 106 AD-MSCs, injected after OPU sessions 1, 2 and 3 (G4). ResultsCharacterization of the MSC-EVs revealed that the size of the particles was similar in the different isolation methods; however, the UF method generated a greater MSC-EV yield. MSC-EVs processed by both methods demonstrated a similar ability to promote cell migration and proliferation in ovarian stromal cells. Considering the higher yield and lower complexity of the UF method, UF-MSC-EVs were used in the in vivo experiment. We evaluated three therapeutic regimens for cows subjected to OPU, noting that the group treated with three MSC-EV injections (G4) maintained oocyte production and increased in vitro embryo production, compared to G1, which presented compromised embryo production following the OPU-induced lesions. ConclusionsMSC-EVs have beneficial effects both on the migration and proliferation of ovarian stromal cells and on the fertility of bovines with follicular puncture injury in vivo.

A developed and validated centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method for rapid quantification of unbound lenvatinib in human plasma

In clinical practice, the determination of unbound drug concentration is very important for dose adjustment and toxicity prediction because only the unbound fraction can achieve a pharmacological effect. A fast, sensitive and accurate analytical method of centrifugal ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry method was developed and applied to allow the quantification of unbound lenvatinib concentration. The application of linear regression analysis was used to examine the effects of centrifugal force, centrifugal time, and protein content on ultrafiltrate volume (Vu). The results indicated that the centrifugal force and centrifugal time have an influence on Vu that is significantly positive (P < 0.05). This developed method with good linearity (r2 = 0.9996), good accuracy (bias % ≤ 2.24 %), good precision (CV % ≤ 7.10 %), and good recovery (95.46 %−106.46 %) was suitable for routine clinical practice and studies. Particularly, the ultrafiltration membrane had no non-specific binding to lenvatinib. The unbound fractions can be separated in just 15 min. This method was applied to quantify clinical samples and to determine the plasma protein binding and unbound fraction of lenvatinib. This study provides a more effective and promising method for determination of unbound lenvatinib. It could be beneficial to measure the unbound concentration of lenvatinib in personalized medicine and therapeutic drug monitoring in routine clinical practice.