Hemoglobin Precipitation: An Index of In Vitro Vasoconstrictive Activities of Methanol Leaf Extracts of Croton megalocarpus Hutch and Lantana camara Linn

The function of innate hemostasis aids the body in bleeding control, preventing the loss of excessive amounts of blood following low-degree injuries. However, injuries of a higher degree may require extrinsic intervention to stop life-threatening blood loss. Astringent agents’ actions result in mechanical constriction of small blood vessels and shrinkage of body tissues, thereby stopping blood loss. This enhances the primary phase of hemostasis, where vasoconstriction is the main mechanism at play during the initial response to injury. The effects of plant extracts on protein precipitation have been linked to blood vessel vasoconstriction. Traditionally, the leaves of Croton megalocarpus Hutch and Lantana camara Linn plants are used by communities living in Makueni County, Kenya, for peripheral bleeding control. However, the effects of extracts of both plants on hemoglobin precipitation have not been evaluated scientifically. In the current study, the activities of methanol extracts of C. megalocarpus (H.) and L. camara (L.) on blood protein precipitation were investigated. The leaves were harvested, cleaned, air-dried, milled, and extracted in absolute methanol before being concentrated into dry powders. A qualitative phytochemical screen revealed the presence of terpenoids, steroids, tannins, phenols, flavonoids, reducing sugars, cardiac glycosides, and carbohydrates in the methanol extract of C. megalocarpus (H.). The methanol extracts of L. camara (L.) contained cardiac glycosides, saponins, tannins, phenols, terpenoids, reducing sugars, and carbohydrates. The hemoglobin precipitation ability of various concentrations of extracts using mice samples was presented as relative astringency following the tannic acid external standard method. Methanol extracts C. megalocarpus (H.) and L. camara (L.) had significantly higher relative astringency compared with the normal control, indicating a protein precipitating activity. The relative astringency observed in both plant extracts is linked to the activity of tannins, phenols, flavonoids, and saponins detected during preliminary phytochemical screening.

Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.

Composition and Protein Precipitation Capacity of Condensed Tannins in Purple Prairie Clover (Dalea purpurea Vent.)

This study aimed to determine the concentration and composition of condensed tannins (CT) in different tissues of purple prairie clover (PPC; Dalea purpurea Vent.) at different maturities and to determine their protein-precipitating capacity. The compositions of CT were elucidated after thiolysis with benzyl mercaptan followed by high-performance liquid-chromatography (HPLC) and 1H–13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The results indicated that PPC flowering heads contained the highest CT concentration. Purple prairie clover CT consisted mainly of epicatechin (EC) and epigallocatechin (EGC) subunits. CT in the leaves were composed of more EC and less EGC than CT in stems and flowering heads at both the early flowering (EF) and late flowering (LF) head stages. The mean degree of polymerization was the highest for CT in stems and increased with maturity. CT isolated from PPC leaves at the early flowering head stage exhibited the greatest biological activity in terms of protein precipitation. Overall, the CT in PPC were predominantly procyanidins and the concentration and composition varied among the plant tissues and with maturity.

Optimized Preparation of Urine Samples from Acute Melioidosis Patients for In-Solution Proteomic Studies using LCMS QTOF or MALDI TOF MS

Introduction: Investigation of urine proteome in patients with acute melioidosis may reveal potential disease markers, from either bacterial or human proteins. We used an in-solution gel-free method instead of 2-DE to detect human and Burkholderia pseudomallei proteins in urine of patients with acute melioidosis. Here, we propose a simpler, economical method for preparing urine samples directly from melioidosis patients, for in-solution proteomic analysis using LCMS-QTOF MS/MS or MALDI-TOF MS/MS. Material and Methods: We adapted an acetone-TCA based protein precipitation method with LCMS-QTOF MS to detect the B. pseudomallei proteins directly from urine of acute melioidosis patients (culture positive and negative). This process involves protein precipitation, desalting, trypsin digestion, and optimization for the mass spectrometry. Results: A total of 3,866 human peptides were detected across 11 urine samples from clinically suspected acute melioidosis patients. Among them were three Burkholderia specific proteins detected in 75% of culture positive samples. Large amounts of acute phase proteins, cell mediated immunity proteins, complement pathway proteins and inflammatory mediators were seen upon gene ontology (GO) annotation and GO enrichment analysis. Conclusions: This simple in-solution sample preparation method can be replicated easily for LCMS/MS-QTOF and MALDI-TOF proteomic analyses, avoiding tedious optimization steps in 2-DE. This method is cost effective and can be done in centres without specialized 2-DE or MS equipment and elutes can be easily transported for analysis and bioinformatics. This is the first study to analyse urine samples directly for B. pseudomallei proteins. Discovery of the entire proteome as a whole is important in leading to biomarker discovery.

Comparison of serum and urinary 5-hydroxyindoleacetic acid as biomarker for neuroendocrine neoplasms

Context Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. Objective To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. Design A retrospective study during 2016 - 2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. Methods Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by two different LC-MS/MS assays with samples prepared either by protein precipitation (PP) or solid phase extraction (SPE). 24-h urine 5HIAA samples (n=33) were analyzed by amperometric LC and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. Results We achieved 5-10 000 nmol/l linearity and ≤2.5% variation with our new serum 5HIAA assay. In ROC analysis the area under curve (AUC) was 85% by serum assays (URL value 123 nmol/l) and 88% by the 24-h urine 5HIAA assay (URL value of 47.1 µmol), respectively. A difference (p<0.001) between patients with active NEN and patients in remission was found by all 5HIAA assays. Conclusion Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.

Quantitative determination of Cpd118, A novel FBPase inhibitor, in dog plasma by HPLC–MS/MS

Aim: A HPLC–MS/MS method was first developed and validated for the quantification of Cpd118, a novel fructose-1, 6-bisphosphatase inhibitor for controlling gluconeogenesis in Type 2 diabetes mellitus. Materials & methods: Cpd118 was extracted from dog plasma following acetonitrile protein precipitation, separated by HPLC on a CAPCELL PAK ADME column (3.5 μm, 2.1 mm × 100 mm) and quantified using negative heated electrospray ion source-MS/MS. Results: Cpd118 was quantified from plasma using the method described above over a linear range of 10–20,000 ng/ml, with interday and intraday assay accuracy from -11.78 to 4.01% and the precision was ≤11.15%. Conclusion: The method was sensitive and selective for the quantification of Cpd118 and was successfully used to the pharmacokinetic and bioavailability study of Cpd118 in dogs.

A Comprehensive Study on Anti-hypertensive properties of Punica granatum (Pomegranate), Cynara scolymus (Artichoke), Coscinium fenestratum (Yellow vine) in Phytopharmacological, Molecular Biology Researches

Hypertension is a turning into a significant danger to the world. In the hunt of lead atoms from plant beginning as a substitute for poisonous engineered drugs, 26 Indian restorative plants and nourishments were screened for their ACE (Angiotensin Converting Enzyme) inhibitory movement. IC50 (half restraint of ACE) estimations of hydroalcoholic unrefined concentrates and division were dictated by a colorimetric technique. Dynamic parts were additionally screened to decide the compound energy, mode, explicitness and instrument of restraint. Normalization was finished by deciding aggregate phenolics and flavonoids as gallic corrosive and quercetin counterparts/mg of concentrate individually. Among 26 unrefined concentrates, Cynara scolymus extricate indicated the best action, IC50 esteem 356.62µg/mL. Pro restraint coming about because of protein precipitation was most noteworthy in Coscinium fenestratum. Lineweaver-Burk plots uncovered a serious method of restraint for Punica granatum ethyl acetic acid derivation part. Divisions of Cynara scolymus were seen as vague inhibitors of ACE. Coscinium fenestratum parts restrained the ACE by Zn2+ particle chelation. Further, in the quest for sheltered and powerful lead atoms from normal sources, (MP) Mucuna pruriens L. (Fabaceae) seeds were used for investigating the antihypertensive potential. Generally it is utilized as diuretic and Hypotensive. Bioassay-guided divisions were used for the separation of dynamic mixes by segment chromatography. IC50 esteem, protein energy and restraint system were resolved. In vivo time and portion subordinate hypotensive examination followed by changes in the MAP (Mean blood vessel pressure) actuated by angiotensin I (3 nmol/kg), angiotensin II (3nmol/kg), and to bradykinin (10nmol/kg) in anesthetized rodents was finished. Plasma and tissue ACE exercises were additionally decided. Phytochemical examination by spectroscopic methods uncovered the nearness of realized mixes like genistein, ursolic corrosive and L-DOPA from the ethyl acetic acid derivation and water part separately. In vitro examination uncovered MP ethyl acetic acid derivation portion (MPEA) and genistein as the most dynamic part (IC50 156.45µg/mL) and compound (IC50 253.81µM) individually. Lineweaver-Burk plots uncovered a non-serious method of hindrance. Expert protein precipitation was the recommended instrument for restraint. The concentrate indicated a time and portion subordinate decline in the MAP.

A Modification of Cell-Lysis for High-Quality Intact RNA of Bacillus Subtilis Extracted From the Culture Under Different Physiological Stages

High quality RNA products from bacterial cells are required for the molecular study. Sample preparation to acquire the high-quality RNA especially the Gram-positive bacteria like Bacillus sp., the model organism, remains a critical burden toward the integration of full molecular downstream analyses although several methods have been proposed including conventional or kit-based protocols. Those techniques were simply developed using the cell samples at certain growth stages unless some molecular studies require RNA samples gathered under different physiological stages of growth and process conditions. Herein, we developed the simple yet effective cell-lysis technique prior to RNA extraction by modifying the commercial kit-based protocols. Bacillus subtilis TL7-3 was used as the model organism in this study. Lysozyme loading (20 mg/mL) as well as the incubation time (30 min) and temperature (37 °C) was responsible for cell lysis and increased RNA concentration in the samples. Invert mixing rather than centrifugation and vortexing prevented RNA damage during protein precipitation by absolute ethanol. This was confirmed by the RNA Integrity Number (RIN) values greater than 8.0 of all RNA extracted from both vegetative cells and endospores of B. subtilis TL7-3. Additionally, absolute ethanol is preferable to our less-than-1-h protocol for protein precipitation as indicated from the higher ratios of A260/A280 and those of A260/A230 of the RNA products than 2.0 and 2.1, respectively. From the findings mentioned above, we successfully developed the modified RNA extraction protocol applicable for the intact cells of Gram-positive bacteria like Bacillus sp. at varied physiological and morphological stages.