Analysis Of Blood Plasma Research Articles

L-plastin levels are associated with mortality during cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.)

Cardiomyopathy syndrome (CMS) is a severe infectious cardiac disease affecting Atlantic salmon (Salmo salar L.). The disease is chief cause of sudden death in Norwegian fish farms and poses serious economical and fish welfare challenges to all salmon-producing countries. Although urgently needed, there are currently no treatments or effective preventive tools available. To achieve this, an in-depth understanding of the biological mechanisms leading to CMS is first required. Proteomic-based blood plasma analysis of inflammatory and immune response proteins recently identified L-plastin as an acute response protein in salmon. Here, we investigated whether L-plastin is associated with piscine myocarditis virus (PMCV) infection and mortality during CMS. By comparing heart and blood plasma samples from surviving and deceased fish from a confirmed CMS outbreak site, we observed that L-plastin expression was elevated in cardiac tissue while circulating free L-plastin levels were reduced in deceased fish. Interestingly, a higher molecular weight band positive for L-plastin was detected in plasma from deceased fish and correlated with L-plastin expression in the heart. In addition, cardiac L-plastin levels correlated with cardiac histopathological changes, mononuclear cell infiltration and necrosis of cardiomyocytes in the heart. Our findings set the base for further investigation of L-plastin as a potential biomarker for mortality risk during PMCV infection and for understanding the course of CMS in farmed salmon.

VALIDASI METODE ANALISIS SENYAWA ETIL p-METOKSISINAMAT DALAM PLASMA DARAH TIKUS PUTIH JANTAN GALUR WISTAR MENGGUNAKAN KROMATOGRAFI CAIR KINERJA TINGGI

Ethyl p-methoxycinnamate (EPMC) isolated from the rhizome of kencur (Kaempferia galanga L) is being developed at STFI. Validation of EPMC analysis in blood plasma using High-Performance Liquid Chromatography (HPLC) needs to be conducted to perform in vivo EPMC analysis. This study aims to validate the EPMC analysis method in blood plasma using HPLC. The HPLC column used is column C18, with methanol : phosphoric acid 70:30 and 80:20 mobile phases, a flow rate of 1.0 mL/min, and a maximum wavelength of 310 nm. The blood plasma used was the blood plasma of white male rats of the Wistar strain. EPMC was added to blood plasma and analyzed using HPLC. Parameter validation used is system suitability test, linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). The results showed that the optimum mobile phase for analyzing EPMS in plasma was methanol : phosphoric acid with a ratio of 70:30. Based on the validation, the linear regression was 0.998. In the accuracy test, the average value range for the percent recovery is 98% - 102%. In the precision test, the average value of the RSD percentage was 4.10% - 68.83%. The LOD value is 2.747 g/mL, and the LOQ value is 9.158 g/mL. Based on this research, it can be concluded that EPMC in blood plasma can be analyzed using KKKT with column C18 and mobile phase methanol : phosphoric acid 70:30.

Metabolomic Profiling of the Blood of Patients with Chronic Consciousness Disorders

The main variants of chronic consciousness disorder (CCD) developing in adverse coma outcome are vegetative state/unresponsive wakefulness syndrome (VS/UWS) and minimal consciousness state (MCS). The aim of the study was to investigate the main differences in metabolomic abnormalities in patients with VS / UWS and MCS, as well as to identify changes in metabolomics depending on sleep or wakefulness phase. Materials and Methods. Untargeted metabolome analysis of blood plasma of 10 patients in VS / UWS (group 1) and 6 patients in MCS (group 2) was performed using reversed-phase and hydrophilic chromatography methods. The underlying conditions of brain injury were TBI (2 in group 1 and 5 in group 2) and hypoxia (8 in group 1 and 1 in group 2). The internal jugular vein was catheterized in all patients, and blood was collected while awake during the daytime for 2 days. Aliquots of pooled plasma samples were purified from protein components and analyzed by high-performance liquid chromatography in two modes: reversed-phase and hydrophilic ones. Mass-spectrometric detection was performed in full ion current scanning mode: registration of positively charged ions in the m/z range from 50 to 1300 a.u. Data were adjusted and normalized using MS-DIAL software ver. 4.70 software; differences were identified using analysis of variance, discriminant and cluster analysis. The data were analyzed and visualized using MetaboAnalyst 5.0 software (https://www.metaboanalyst.ca). Results. Four major metabolites (at VIP > 0.5), which content was most modulated depending on the study group, were identified including 4 (m/z 124.0867, Rt = 17.67, p < 0.01), 33 (m/z 782.5722, Rt = 17.69, p < 0.01), 6 (m/z 125.0904, Rt = 18.43, p < 0.01) and 1 (m/z 463.2304, Rt = 15.78, p < 0.01), with no significant differences between daytime and nighttime blood samples. Significant quantitative differences were shown for three metabolites in the groups, 14 (m/z 162.1126, Rt = 10.28, p < 0.01), 35 (m/z 780.5483, Rt = 7.65, p < 0.01), and 41 (m/z 806.5649, Rt = 7.58, p < 0.01), and four metabolites when comparing the daytime and nighttime samples: 14 (m/z 162.1126, Rt = 10.28, p = 0.0201), 35 (m/z 780.5483, Rt = 7.65, p < 0.01), 41 (m/z 806.5649, Rt = 7.58, p < 0.01), and 48 (m/z 848.5354, Rt = 7.65, p < 0.01). Conclusion. Untargeted metabolomic analysis confirmed the hypothesis of likely significant quantitative and qualitative differences in metabolite composition depending on the type of CCD and circadian rhythm. The study established a set of metabolites that are potential biomarkers for differential diagnosis of VS/UWS and MCS including 4, 33, 6, 1 (in the experiment on the reversed-phase column) and 14, 35, 41, 48 (in the experiment on the hydrophilic column), based on their significant contribution to intergroup and intragroup differences. Further studies will be aimed to characterize the identified metabolites.

Especiação de estanho no plasma sanguíneo de trabalhadores expostos ocupacionalmente em uma indústria de processamento de minério de cassiterita

ABSTRACT Mining is a high-risk activity due to its dangerous processes. Tin (Sn) is obtained from cassiterite ore and mining activities expose workers to the metal. Chronic exposure to Sn may cause pneumoconiosis, gastrointestinal and hematological effects, among others. This work aimed to assess the exposure of workers to tin in a cassiterite ore processing industry, using the speciation analysis in blood plasma. Twelve subjects donated the blood samples; six were occupationally exposed to Sn. Size exclusion chromatography separated proteins in blood plasma; a graphite furnace atomic absorption spectrometer determined total tin in the plasma and eluted fractions, while SDS-PAGE determined molecular masses of proteins. Tin levels in the workers’ plasma were four times higher than in the reference individuals. After fractionation, the metal only appeared in the total inclusion volume, not being possible to confirm the binding of tin to proteins, which certainly modifies their functions and impair workers’ health. Despite that, the work process needs to change since Sn levels in the workers’ plasma pointed to metal exposure. Further works are necessary to clarify whether the metal is free or bound to small proteins in blood plasma and understand the true impact of tin on workers’ health.

Continued Virus-Specific Antibody-Secreting Cell Production, Avidity Maturation and B Cell Evolution in Patients Hospitalized with COVID-19.

Understanding the development and sustainability of the virus-specific protective immune response to infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains incomplete with respect to the appearance and disappearance of virus-specific antibody-secreting cells (ASCs) in circulation. Therefore, we performed cross-sectional and longitudinal analyses of peripheral blood mononuclear cells and plasma collected from 55 hospitalized patients up to 4 months after onset of COVID-19 symptoms. Spike (S)- and nucleocapsid (N)-specific IgM and IgG ASCs appeared within 2 weeks accompanied by flow cytometry increases in double negative plasmablasts consistent with a rapid extrafollicular B cell response. Total and virus-specific IgM and IgG ASCs peaked at 3-4 weeks and were still being produced at 3-4 months accompanied by increasing antibody avidity consistent with a slower germinal center B cell response. N-specific ASCs were produced for longer than S-specific ASCs and avidity maturation was greater for antibody to N than S. Patients with more severe disease produced more S-specific IgM and IgG ASCs than those with mild disease and had higher levels of N- and S-specific antibody. Women had more B cells in circulation than men and produced more S-specific IgA and IgG and N-specific IgG ASCs. Flow cytometry analysis of B cell phenotypes showed an increase in circulating B cells at 4-6 weeks with decreased percentages of switched and unswitched memory B cells. These data indicate ongoing antigen-specific stimulation, maturation, and production of ASCs for several months after onset of symptoms in patients hospitalized with COVID-19.

STRUCTURAL-FUNCTIONAL AND MOLECULAR DIAGNOSTIC PARALLELS IN ARTERIAL HYPERTENSION IN ELDERLY PATIENTS

Abstract: Relevance. Among cardiovascular diseases arterial hypertension (AH) in elderly people over 60 years old is the main risk factor for stroke, heart failure, terminal renal failure and lethal outcome. Today hypertension is defined as accelerated aging of the arterial component of the cardiovascular system with developing of remodeling processes due to age. Studies of a molecular phenotype of hypertension in elderly patients with obtaining molecular profiles of biological samples determining the clinical course of hypertension and its classification characteristics are topical nowadays. The aim of the study is to search correlations between structural, functional and molecular diagnostic parameters in patients with AH of Grade 2 of a moderate and high risk. Methods and materials: The study involved patients over 60 years old with a diagnosis of Grade 2, moderate and high risk hypertension (Group 2, n=200), taking into consideration the criteria of inclusion and non-inclusion into the study. The control group (KG, Group 1) included 30 elderly people with no hypertension. Central blood pressure (BP) in the aorta and pressure on the brachial artery were measured, indicators of daily blood pressure monitoring, structural and functional parameters of the heart were evaluated on the basis of echocardiography. There was fulfilled a qualitative proteomic analysis of blood plasma based on time–of–flight mass spectrometry with a laser desorption and ionization and quantitative assessment of the level of proteins in the blood with the help of enzyme immunoassay. Results: There was revealed an increase of the blood concentration of MPO, apoD, HIF1α, TGFβ1, hyaluronidase 1, kininogen 1, fibulin 5 and their significant correlations with LVMM and ACC. Conclusions: There was determined diagnostic molecular profile of blood of elderly patients with AH of Grade 2 and confirmed the involvement of proteins in myocardial and vascular remodeling in elderly patients with AH.

High-recovery sorting of cancer cells from whole blood via periodic-focusing inertial microchip.

Inertial microfluidic devices continue to show promise for label-free separation of cells from liquid biopsies and other biological samples. Serpentine-channel microfluidic devices capitalizing on inertial forces such as Dean flow have been demonstrated for cell separation, but are limited in performance due to the magnitude of the inertial lift and drag force gradients across the separation channel. We have developed a new flow design that uses periodic channel contractions to enhance the magnitude of the force gradient. Separation recover was 97% with the final sorter output consisting of 78% target cells. Separation efficiency was 87% for whole blood, which could be increased to 97% if the sample was diluted prior to sorting. The enrichment of cancer cells was over 1000-fold, and sorted cancer cells maintained a viability of 93.8% for 96 hours after sorting. In the analysis of blood plasma, breast cancer cells from a clinical patient were enriched 20×. The incorporation of periodic channel contractions in a Dean flow circuit resulted in an increase in Dean flow gradient according to simulation, resulting in sorting of small-diameter cancer cells in blood samples.

Optimization of injection molded polymer lab-on-a-chip for acoustic blood plasma separation using virtual design of experiment

This study presents the development towards the optimization and manufacturing of a novel cost-effective fully polymer-based ultrasound actuated chip as a Point-of-care (POC) diagnostic platform for blood plasma separation and analysis. The chip was designed following multi-physic functional simulation to obtain optimal dimensions with respect to blood separation enhanced capacity. Initial dimensional guidelines showed most favorable performance to be achieved with design dimensions of 150 µm height, 375 µm width and 34 mm length. The chip encompasses a micro-fluidic channel with a relatively long length compared to the height and the width. As such, main manufacturability challenge is represented by the capability to maintain the channel dimensional conformance throughout the entire length. In order to tackle the challenge and optimize the results, developing a feedback loop was initiated for the device process development where the data of the preliminary micro-injection molding (µIM) production was exploited to implement a simulation-based full-factorial virtual design of experiment (DOE) for all the four materials of interest: polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), polycarbonate (PC) and polystyrene (PS). The four essential µIM parameters namely, mold temperature, melt temperature, injection velocity and packing pressure were used as factors. Overall, simulations were performed based on the virtual DOE inputs for the four aforementioned materials. The input data for the utilized process parameters in the virtual DOE was provided by our first production batch. Ultimately, in the case of PMMA, parts were molded and characterized based on the process settings that had demonstrated the lowest linear shrinkage in our entire virtual DOE responses. For the linear shrinkage of these parts, a respective width and height difference of 1% and 3.3% between the simulations and production results were obtained. Moreover, as a secondary study sink marks were also inspected and minimized through simulations. Initially, sink marks as deep as 80 µm were spotted on the chip. After the use of optimization cycle the value decreased to as low as 10 µm. The results confirm the positive impact of the virtual experiments for future rapid assessment of potential new chip designs.

The clinical efficacy of pentoxifylline and l-glutamine on ischemia and reperfusion injury in cattle with displaced abomasum: a longitudinal study.

This study aimed to assess the clinical efficacy of pentoxifylline (PTX) and L-glutamine (L-Gln) treatment on ischemia and reperfusion (I/R) injury in the abomasal tissue, acute phase response (APR), oxidative stress (OS), cytokine response, hemostatic, and coagulation disorders in the 96-h period before and after surgery in displaced abomasum (DA) cases. The study sample consisted of 48 dairy cows with DA that were categorized into four groups as group S (Sham group) (9 Left displaced abomasum (LDA)+3 Right displaced abomasum (RDA), group P (PTX) (10 LDA+2 RDA), group G (L-Gln) (10 LDA+2 RDA), and group P+G (PTX+L-Gln) (10 LDA+2 RDA). Acute-phase protein (Haptoglobin), oxidative stress indicators (malondialdehyde, nitric oxide, and glutathione), cytokines (tumor necrosis factor (TNF)-α and interleukin-1β (IL-1β), coagulation factors (D-Dimer, Antithrombin (ATIII), Thrombin-antithrombin complex, Plasminogen activator inhibitor-1), and enzyme activities (lactate dehydrogenase, gamma- -glutamyl transferase, sorbitol dehydrogenase, glutamate dehydrogenase, adenosine deaminase, myeloperoxidase, and creatine phosphokinase) in blood serum samples and coagulometric analyses of blood plasma were performed in samples taken before the operation and at 30 and 60 min and 2, 5, 10, 24, 48, 72, and 96 h after the operation. In DA cases, while post-operative treatment procedures with PTX and L-Gln were effective in decreasing APR and OS, these were ineffective in prohibiting the inflammatory response coordinated by cytokines. For the treatment and prevention of I/R injury in the DA cases, PTX and L-Gln procedures hold promise with their effects on APR, OS, and hemostatic dysfunction. Additional treatment procedures are required for the suppression of inflammatory response, and the effectiveness of preconditioning treatment may be evaluated.

BaZrO3 based non enzymatic single component single step ceramic electrochemical sensor for the picomolar detection of dopamine

BaZrO3 perovskite crystallites were synthesized in a single step using a calcination free room temperature co precipitation method. Crystallite size, presence of metal oxygen bond, thermal stability, defect structure, surface morphology were studied using structural, thermal and microscopic investigations. This material was used for the modification of carbon paste electrode (CPE) for the fabrication of an advanced sensor. The modified electrode exhibited lower resistance and better charge transfer compared to an unmodified electrode. The electrochemical characteristics investigated using CV and DPV showed high current sensitivity towards the detection of dopamine (DA). The modified electrode achieved a remarkable low concentration of 5 pM detection limit level for the detection of DA. Limit of detection for co existing molecules of uric acid (UA) and ascorbic acid (AA) were 500 pM and 500 nM respectively. The developed sensor could quantify the DA in the presence of uric acid (UA) and ascorbic acid (AA). Mixed sample analysis, interference studies, stability analysis etc prove the competence of the developed sensor. Real sample analysis in blood plasma and urine marked the practical utility of the developed sensor.

Comparative study of stress responses, laterality and familiarity recognition between albino and pigmented fish

Oculocutaneous albinism is the result of a combination of homozygous recessive mutations that block the synthesis of the tyrosine and melatonin hormones. This disability is associated with physiological limitations, e.g., visual impairment expressed by lower visual acuity and movement perception, and eventually leads to acrophobia and/or photophobia, suggesting a potentially higher stress level associated with the behavioral responses of individuals with albinism to external stimuli compared to their pigmented conspecifics. However, in fish, differences in behavioral and/or physiological responses and stress levels between these phenotypes have been poorly documented. While acoustic perception of albino individuals is well known, the use of olfactory sensors for social communication, e.g., for the preference for familiar conspecifics, remains poorly understood. We performed two laboratory experiments with albino and pigmented European catfish Silurus glanis to observe: i) their behavioral and physiological responses to short-term stress induced by a combination of air exposure and novel environmental stressors and ii) their ability to use odor keys to recognize of familiar conspecifics and the influence of lateralization on this preference. In response to stress stimuli, albino fish showed higher movement activities and ventilatory frequencies and more often changed their swimming directions compared to their pigmented conspecifics. Blood plasma analysis showed significantly higher values of stress-, deprivation-, and emotional arousal-associated substances, e.g., glucose and lactate, as well as of substances released during intensive muscle activity of hyperventilation and tissue hypoxia, e.g., hemoglobin, mean corpuscular hemoglobin, erythrocytes, and neutrophil granulocytes. A preference test between environments with and without scented water showed the preference by both albino and pigmented catfish for environments with scent of familiar conspecifics, and both groups of fish displayed left-side lateralization associated with the observation of conspecifics and group coordination. The results tended to show higher physiological and behavioral responses of albinos to stress stimuli compared to the responses of their pigmented conspecifics, but the uses of olfactory sensors and lateralization were not differentiated between the two groups.

Flavonoids derived from Anemarrhenae Rhizoma ameliorate inflammation of benign prostatic hyperplasia via modulating COX/LOX pathways

Ethnopharmacological relevanceFlavonoids are the main components of the traditional Chinese medicine Anemarrhenae Rhizoma (dried rhizome of Anemarrhena asphodeloides Bge.), which has been reported to possess activity against inflammation and tumor. Aim of studyRegulation of the arachidonic acid (AA) cascade through cyclooxygenase (COX) and lipoxygenase (LOX) represent the two major pathways to treat inflammatory of benign prostatic hyperplasia (BPH). In this study, Anemarrhenae Rhizoma flavonoids and its main compounds (mangiferin, neomangiferin and isomangiferin) were investigated for effects on AA metabolism. MethodsUltra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to monitor AA metabolites in BPH rats and in PC-3 cells. COX-2 and 5-LOX protein and mRNA levels were measured by Western blot and qPCR, respectively, along with histopathological assessment of prostate tissues. ResultsTreatment with flavonoids significantly ameliorated BPH-associated prostate inflammation and inhibited the expression of COX-2 and 5-LOX at the protein and mRNA levels. Quantitative metabolomic analysis of blood plasma showed flavonoids treatment decreased AA levels and its metabolites associated with the COX and LOX pathways. Further exploration of the flavonoid compounds mangiferin, neomangiferin and isomangiferin showed they inhibited AA metabolism to varying degrees in PC-3 cell cultures. ConclusionAnemarrhenae Rhizoma flavonoids act to inhibit BPH-related inflammation in vivo and in vitro by targeting AA metabolism and interfering with COX and LOX pathways. The identification of mangiferin, neomangiferin and isomangiferin as anti-inflammatory components suggests flavonoids interventions represent a promising therapeutic approach for BPH.

Potenciál spektroskopie v diagnostice hepatocelulárního karcinomu – pilotní studie

Summary: None of the bio­markers studied so far in the HCC area has yielded higher sensitivity and specificity in the early-stage dia­gnosis than the liver ultrasonography examination. There is an urgent clinical need for establishing a laboratory marker for HCC that meets the requirements for high sensitivity and specificity for the screening and early dia­gnosis of at-risk patients. As a variety of pathological processes, including carcinogenesis, may cause changes in both the concentration and the structure and spatial arrangement of body bio­molecules, the spectroscopic analysis of blood-based derivatives appears to be an appropriate tool for the early detection thereof. In our research, the focus is on the identification of novel bio­markers in blood plasma, which would exhibit sufficient sensitivity and specificity to detect early and potentially curable HCC stages, and which would be potentially useful for routine screening of this disease in well-defined at-risk groups. For this purpose, we utilised a unique combination of two chiroptical methods – electronic circular dichroism (ECD) and Raman optical activity (ROA) – supplemented by non-polarised variants – infrared (IR) absorption and Raman spectroscopy. Methods: Blood plasma of 18 selected patients with liver cirrhosis, 8 of which also suffered from HCC, was analysed by a combination of ECD, ROA, IR and Raman spectroscopy. Results: The obtained spectral data were processed by a multivariate statistical evaluation using principal component analysis (PCA) and linear discriminant analysis (LDA). The visualisation of the LDA results showed the separation of the two monitored groups with only a slight overlap. Based on the spectral analysis within this preliminary study, sensitivity and specificity for the discrimination between cirrhotic individuals with and without HCC reached 88% and 90% after leave-one-out cross validation, respectively. The area under the ROC curve of 0.975 proved high reliability of the established model. Conclusion: Based on our findings, the combination of advanced spectroscopic methods for the analysis of blood plasma might be a promising tool in HCC dia­gnosis and potentially in the screening thereof. Key words: hepatocellular carcinoma – blood plasma – spectroscopy – cirrhosis

Vascular region-specific changes in arterial tone in rats with type 2 diabetes mellitus: Opposite responses of mesenteric and femoral arteries to acetylcholine and 5-hydroxytryptamine

AimsType 2 diabetes mellitus (T2DM) ranks in the top 10 causes of mortality worldwide. The key factor of T2DM vascular complications is endothelial dysfunction. It is characterized by the vessels motor activity disruption and endothelium-derived factors imbalance. The blood vessels morphological and molecular heterogeneity greatly affects the changes occurring in T2DM. Therefore, we conducted a comparative study of vascular bed changes occurring in T2DM. Main methodsMale Wistar rats were fed a high-fat diet for 20 weeks, followed by a single streptozotocin injection (20 mg/kg). T2DM was confirmed with an oral glucose tolerance test. Key findingsA dose-dependent contraction study showed an increase in third-order mesenteric arterioles response to serotonin but not to phenylephrine. These vessels also exhibited a decrease in acetylcholine-dependent relaxation and an increase in guanylate cyclase function. At the same time, the femoral arteries showed a tendency for increased acetylcholine-dependent relaxation. The blood plasma analysis revealed low bioavailable nitric oxide and high levels of endothelin-1 and ROS. SignificanceThis knowledge, in conjunction with the features of the T2DM course, can allow further targeted approaches development for the prevention and treatment of vascular complications occurring in the disease.

2D-DIGE proteomic analysis of blood plasma reveals changes in immune- and stress-associated proteins following hormonal stimulation of carp males

In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2 <−1.2-fold change, P < 0.05) in the abundance of 30 spots (14 up- and 16 downregulated) and 44 spots (28 up- and 16 downregulated) upon CPE and Ovopel treatment, respectively. The most significantly affected pathways were acute phase response signaling, the coagulation system, LXR/RXR and FXR/RXR activation; however, there were different sets of proteins in Ovopel- and CPE-treated males. The majority of differentially abundant proteins were involved in the regulation of the immune defense response, the response to stress, and complement activation. Moreover hormonal stimulation with CPE markedly increased the bactericidal activity of blood and both preparations caused profound changes in gene expression in hematopoietic organs. This work is important in understanding the biological processes behind the protein-based response to hormonal stimulation of sperm production in fish.

Co-Spray Dried Nafamostat Mesylate with Lecithin and Mannitol as Respirable Microparticles for Targeted Pulmonary Delivery: Pharmacokinetics and Lung Distribution in Rats.

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution of NFM, administered via intravenous and intratracheal routes, were determined using high performance liquid chromatography analysis of blood plasma, lung lumen using bronchoalveolar lavage fluid, and lung tissue. Intratracheal administration had higher drug delivery and longer residual time in the lung lumen and tissue, which are the main sites of action, than intravenous administration. We confirmed the effect of lecithin as a stabilizer through an ex vivo stability test. Lecithin acts as an inhibitor of carboxylesterase and delays NFM decomposition. We prepared inhalable microparticles with NFM, lecithin, and mannitol via the co-spray method. The formulation prepared using an NFM:lecithin:mannitol ratio of 1:1:100 had a small particle size and excellent aerodynamic performance. Spray dried microparticles containing NFM, lecithin, and mannitol (1:1:100) had the longest residual time in the lung tissue. In conclusion, NFM-inhalable microparticles were prepared and confirmed to be delivered into the respiratory tract, such as lung lumen and lung tissue, through in vitro and in vivo evaluations.

Verification of Lactobacillus brevis tolerance to simulated gastric juice and the potential effects of postbiotic gamma-aminobutyric acid in streptozotocin-induced diabetic mice

The therapeutic effect of gamma-aminobutyric acid (GABA) on diabetes was spread as one of the alarming epidemics worldwide. The study aims to investigate the function of Lactobacillus brevis KLDS1.0727 and KLDS1.0373 strains as glutamic acid decarboxylase 65 (GAD65) carriers capable of generating GABA by comparing in vitro free and freeze-dried models and GABA intervention in vivo. PCR amplification of gad and in vitro i.e., (growth rate, viability at different pH, bile tolerance, and survivability in simulated gastric juice) were performed. In vivo experiments were conducted in 7 groups of C57BL/6J mice. Each group was injected with streptozotocin (ContSTZ, INSSTZ, LAC1STZ, LAC1MFDSTZ, LAC2STZ, LAC2MFDSTZ) daily except for the control (Cont). One group was injected with insulin (INSSTZ). The body weight and hyperglycemia in the blood were assessed weekly, post-euthanasia blood plasma parameters, insulin, and histological examination were evaluated. Results indicated L. brevis strains demonstrated a great tolerance to bile and simulated gastric juice in vitro (P<0.05). ContSTZ had the highest average glucose level (6.84±6.46) mmol/L while INSSTZ expressed dramatically decreed in glucose level and displayed a significant decline in the average of weekly blood glucose (−5.74±3.08) mmol/L. The lowest body weight (ContSTZ) was (19.30±0.25) g. Based on the blood plasma analysis, L. brevis strains improved good cholesterol properties, liver and kidney functions, where most of these parameters fall within the average the reference range and prevent the development of symptoms of type 1 diabetes in vivo. As recommended, L. brevis should be commonly distributed as a postbiotic GABA in pharmaceutical and nutritional applications.

INFLUENCE OF BASIL OIL AS AN ESSENTIAL OIL ON BUFFALO CALVES PERFORMANCE, DIGESTIBILITY, HAEMOBIOCHEMICAL PROFILE AND RUMEN FERMENTATION INDICATORS

This work was fulfilled to assess the effect of supplementation diets with Basil oil in buffalo calves on growth performance criteria, rumen fermentation properties and certain biochemical indices. A total number of 18 buffalo calves of (4-6) months age and 141.29 kg average body weight were classified randomly into three equal groups (6 animals each): Animals of T1 (control) were fed on basal diet and those of T2 and T3 (treated) were fed as T1 plus 2 and 4 ml basil oil (BO) per head daily, respectively. This experiment was expanded for fifteen weeks. Blood plasma and rumen liquor Samples were obtained from all experimental animals in all groups at the finishing of study for detecting certain blood biochemical parameters and rumen fermentations properties. Results showed that the groups supplemented by basil oil (T2 and T3) recorded significant improvement (P≤0.05) in DM intake, digestibility, growth performance and best feed conversion ratio compared with control group (T1). Also, treated groups (T2 and T3) recorded non observable (P≥0.05) effects in TVFA's, total protozoa count (TPC) and ammonia concentration compared with control group. Blood plasma analysis in supplemented groups (T2 and T3) showed significant improvement (P≤0.05) in protein profile (total protein and albumin), non-significant decrease in urea, creatinine, triglycerides, liver function enzymes levels in comparing with control group. The results collected from this study proposed that buffalo calves fed diets supplemented with basil oil as an essential oil, showed greater total weight gains, daily weight gains, and feed conversion rates with better rumen fermentation parameters.

Abstract 459: Lipidomic analysis of extracellular vesicles and its potential for the identification of body fluid-based biomarkers for breast cancer diagnosis

Abstract Background: Tumor-derived Extracellular Vesicles (EVs) are found in body fluids of cancer patients. These EVs include exosomes and microvesicles, which play an important role in tumor development and metastasis. Although EVs have great potential as liquid biopsies for early detection of breast cancer, the lipid composition of breast cancer-derived EVs is unknown. Here, we studied the lipid composition of EVs and its potential for the identification of body fluid-based biomarkers for breast cancer diagnosis. Methods: The lipid composition of EVs and their parental cells were evaluated for nine breast cancer and two non-cancerous mammary cell lines. EVs were isolated by size-exclusion chromatography (SEC). Furthermore, ten blood plasma samples were studied from women diagnosed with primary breast cancer before surgery, nine samples from patients with progressive metastatic breast cancer and ten samples from healthy women who neither had pre-existing medical conditions nor infections at the time the samples were collected or in the previous weeks. EVs were obtained from blood plasma samples by a combination of density gradient ultracentrifugation and SEC. Lipids were extracted from EVs and analyzed by reverse-phase liquid chromatography mass-spectrometry (LC-MS). Results: We found that EVs produced by breast cancer cell lines were enriched in sphingomyelins and ceramides when compared to their parental cells. Furthermore, phospholipids such as phosphatidylcholine and phosphatidylethanolamine were found to be abundant in these EVs. Multivariate analyses showed that EVs released by breast cancer cells can be distinguished from those released by non-cancerous cells based on their phospholipid and sphingolipid composition (AUCROC=0.99, p&amp;lt;0.001). A key finding of this study is that we also found significant differences in the phospholipid and sphingolipid composition between EVs found in blood plasma from breast cancer patients and healthy volunteers (AUCROC=0.99, p&amp;lt;0.001). Importantly with respect to the practical applications of this study for early diagnosis of breast cancer, these differences in EV's lipid content were also significant between EVs from primary breast cancer patients and healthy volunteers (AUCROC=0.99, p&amp;lt;0.001). Annotation of specific lipid species are currently being carried out by MS/MS analysis. Conclusion: Our findings demonstrate the potential of the lipid content of cancer-derived EVs for the identification of lipid biomarkers for breast cancer diagnosis and its use for the development of non-invasive (or minimally invasive) diagnostic methods based on blood plasma analysis. Citation Format: Erika Dorado, M Luisa Doria, Anika Nagelkerke, James S McKenzie, Stefania Maneta-Stavrakaki, Charlotte Ion, Thomas Whittaker, Jeremy Nicholson, Molly M Stevens, R Charles Coombes, Zoltan Takats. Lipidomic analysis of extracellular vesicles and its potential for the identification of body fluid-based biomarkers for breast cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 459.

Can the Intake of a Synthetic Tryptamine be Detected Only by Blood Plasma Analysis? A Clinical Toxicology Case Involving 4-HO-MET.

Tryptamines represent a group of hallucinogenic new psychoactive substances with increasing prevalence. Unfortunately, only limited data concerning their toxicology and bioanalysis are available as tryptamines are not included in routine screening procedures in many laboratories. In order to expand the current knowledge, we report a non-fatal clinical toxicology case involving the synthetic tryptamine 4-HO-MET (4-hydroxy-N-methyl-N-ethyl-tryptamine, 3-{2-[ethyl(methyl)amino]ethyl}-1H-indol-4-ol, metocin or methylcybin). As only blood of the intoxicated patient was available, our systematic blood plasma screening approaches based on gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) coupled to low-resolution linear ion trap mass spectrometry (ITMSn) or high-resolution tandem mass spectrometry (HRMS-MS) were conducted. The ingestion of the synthetic tryptamine 4-HO-MET could be revealed by blood plasma analysis using both LC-based systematic screening approaches. However, 4-HO-MET was not detected by GC-MS. Furthermore, the detection of metabolites, which may be used to confirm an intake of the parent compound 4-HO-MET, was only successful using LC-HRMS-MS most probably due to its increased sensitivity compared to LC-ITMSn. A total of four metabolites were detected in blood, including N-demethyl-, oxo- and hydroxy-4-HO-MET, as well as the N-oxide. Finally, LC-HRMS-MS analysis revealed a plasma concentration of 193 ng/mL for 4-HO-MET using the standard addition method. The presented data may help clinical and forensic toxicologists with the interpretation of future cases involving synthetic tryptamines, especially if only blood samples are available.