Serum Components Research Articles

Effects of Feeding Low Protein Diets on Serum and Faeces Parameters in Weaned Piglets

Abstract This study was conducted to determine the effects of a low-protein diet supplemented with synthetic amino acids on the biochemical parameters in the blood serum, the indicators of fermentation processes, and nitrogen excretion in 12 crossbred piglets. The piglets (weaned at 28 days of age) were divided into two groups with 6 piglets each. The control group had an initial average body weight of 8.8 ± 0.6 kg and the experimental group with an average initial body weight of 8.6 ± 0.7 kg. The control diet contained 210.8 g.kg−1 crude protein and the experimental diet contained 186.4 g.kg−1. The experimental diet was supplemented with lysine, methionine and threonine to achieve a more ideal amino acid pattern. The blood collections from the sinus ophthalmicus for the determination of the biochemical parameters were performed 4 times at weekly intervals in the control and experimental groups 4—5 hours after feeding. The faeces were taken from the rectum at the end of the study period. The decrease in the dietary crude protein content of the experimental group was manifested by a significant decrease of the blood urea level (2.61 mmol.l−1 average concentration) compared to the control groups (4.21 mmol.l−1 average concentration) (P < 0.001). The other serum component concentrations (total protein, albumin, glucose, cholesterol, total lipids and selected enzymes) showed no significant statistical changes between the control and experimental groups. The results of the fermentation process analysis indicated that the butyrate concentration decreased (P = 0.0017) and the pH increased (P = 0.0180) in the experimental group compared to the control group. The levels of crude protein and ammonia in the faeces of experimental animals were significantly lower (P < 0.001) in comparison with those in the control animals.

A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy

Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

Extraction-free spectrophotometric assay of the antitussive drug pentoxyverine citrate using sulfonephthalein dyes.

Pentoxyverine citrate (PEN-citrate) is an antitussive (cough suppressant) drug used for cough associated with illnesses like common cold. In this work, PEN-citrate is quantified by applying a simple, direct and accurate spectrophotometric method in pure form, pharmaceutical formulation (Cabella®, 2.13 mg/mL) and human serum samples. The formation of a stable yellow ion-pair with sulfonephthalein dyes; bromocresol green (BCG), bromophenol blue (BPB), bromothymol blue (BTB), bromocresol purple (BCP), bromochlorophenol blue (BChPB) and bromoxylenol blue (BXB), in three nonpolar solvents (chloroform, dichloromethane, acetonitrile) is used as the basis for this method. This is the first assay method reported for the quantification of PEN-citrate using the sulfonephthaleins as coloring agents. Diverse parameters were investigated in order to optimize the calibration curve conditions. The strategy was validated with respect to linearity range, precision, accuracy, specificity, robustness and limits of detection (LOD) and quantification (LOQ). In addition, solvents of different polarities were utilized to investigate the color reaction, light absorption and to allow for increasing the method sensitivity. Beer's law is obeyed over a wide concentration range (up to 42.05 μg/mL in case of BTB method). LOD and LOQ values reached 0.22 and 0.72 μg/mL, respectively, upon using BChPB. The relative standard deviation (%RSD) was ≤1.91% while correlation coefficient values (r) were ≥ 0.9974. High molar absorptivity values and low values of Sandell's sensitivity were obtained indicating that the proposed methods are highly sensitive. The validated methods were applied to the analysis of PEN-citrate in the dosage form and human serum samples where the drug was successfully resolved from the pharmaceutical additives and serum components with recoveries ≥98.98%.

Validated LC-MS/MS method for simultaneous quantification of seven components of Naodesheng in rat serum after oral administration and its application to a pharmacokinetic study

A simple, precise and reliable LC-MS/MS method was developed and validated for simultaneous quantification of vitexin, notoginsenoside R1, hydroxysafflor yellow A, ginsenoside Rd, puerarin, daidzein and senkyunolide I as components of Naodesheng (NDS) in rat serum. The Linearity ranges in rat serum were 0.045–4.5 μg/mL for vitexin, 0.0476–4.76 μg/mL for notoginsenoside R1, 0.0422–4.22 μg/mL for hydroxysafflor yellow A, 0.0426–4.26 μg/mL for ginsenoside Rd, 0.0436–4.36 μg/mL for puerarin, 0.026–2.6 μg/mL for daidzein, and 0.05–5 μg/mL for senkyunolide I, with the correlation coefficients greater than 0.99. The established method was validated in terms of intra- and inter-day precision and accuracy, recovery, matrix effect and stability. Furthermore, the method was successfully applied for pharmacokinetic study of these seven components in rat serum after oral administration of NDS.

The Relationship between Serum Thyrotrophic and Components of Metabolic Syndrome

Background and objective: Metabolic syndrome (Meets) is a cluster of obesity, hyperglycemia, dyslipidemia and hypertension (HTN). Thyroid hormones play an important role in regulating energy homeostasis, carbohydrate, lipids and protein metabolism. Therefore the present study was an effort to investigate the influence of TSH levels in each component of patients with Meets in a population of Saudi Arabia. Design: We analyzed retrospectively 656 participants with MetS whom are between the ages 20 to 98 years. All patients were from the population of the Primary health centre at King Fahad Armed Forces Hospital, Jeddah, Saudi Arabia. All data were collected on the basis of a review of electronic medical data. Patient who are pregnant were excluded. The reference range values of TSH 0.22-4.2 MIU/L, Free T4 12.0-22.0 pmol/L. TSH was divided into three groups; <1.5, 1.5-2.5 and >2.5-4.2. Metabolic risk factors were defined using the 2006 IDF criteria that define elevated triglyceride as ≥150 mg/dL (≥1.7 mmol/L) and reduced high density lipoprotein cholesterol (HDL) as <40 mg/dL (<1.03 mmol/L) for male and as <50 mg/dL (<1.29 mmol/L) for female. Elevated blood pressure was defined when the systolic blood pressure was ≥130 mm Hg and/or diastolic blood pressure was ≥85 mm Hg in addition to receiving any medication for HTN. Abnormal glucose metabolism was con¬sidered when HbA1c (≥5.7) or when patients were known to have type 2 diabetes mellitus (T2DM). The total number of cohort was separated on basis of age values into four groups: <40 years, 40-49 years, 50-59 years and ≥60 years. Results: 656 subjects with MetS were included. There were 86 (13.1% ) male and 570 (86.9%) were female with mean age 55.6 ±12.7 with mean body mass index 32.8 ± 7.2 kg/m2 . HbA1c>5.6 or T2DM, hypertension, triglyceride (≥1.7 mmol/l) and low HDL were present in 94 (14.3%), 354 (54.0%), 328 (50.2%) and 487 (74.2%) respectively. The mean TSH and FT4 values were 2.1 ±1.0 mIU/l and 15.4 ±3.1 pmol/l respectively. Patients with TSH (>2.5-4.2) were non-significantly younger and have significantly higher BMI compared to patients with TSH (<1.5) or TSH (1.5-2.5), 50.9 ±12.4 vs. 53.5 ±12.7 vs. 53.4 ±13.0 respectively, p=0.05 and 33.6 ± 8.2 vs. 31.7 ± 6.1 vs. 33.2 ± 7.0 respectively, p=0.01. Females compared to males were non-significantly predominant in patients with TSH (>2.5-4.2) compared to patients with TSH (<1.5) or TSH (1.5-2.5), 87.3 vs. 12.7%, 89.4 vs. 10.6% and 84.0 vs. 16.0%, p=0.2. Cases with HbA1c>5.6 or T2DM were significantly more prevalent in patients with TSH (>2.5-4.2) compared to TSH (<1.5) or TSH (1.5-2.5), 18.8% vs. 13.9% vs. 10.0 respectively, p=0.03. Cases with HTN were significantly less prevalent in patients with TSH (>2.5-4.2) compared to TSH (<1.5) or TSH (1.5-2.5), 46.3% vs. 54.3% vs. 61.6 respectively, p=0.005. Cases with serum triglyceride (≥1.7 mmol/l) were significantly more prevalent in patients with TSH (>2.5-4.2) compared to TSH (<1.5) or TSH (1.5-2.5), 57.6% vs. 44.9% vs. 47.2 respectively, p=0.02. Cases with low HDL were significantly more prevalent in patients with TSH (>2.5-4.2) compared to TSH (<1.5) or TSH (1.5-2.5), 85.1% vs. 74.9% vs. 71.1 respectively, p=0.002. Higher prevalence of HbA1c>5.6 or T2DM, low HDL , triglyceride (≥1.7 mmol/l) and HTN in patients with age 40-59 years compared to <40 years or more than 60 years, p<0.0001, p=0.4, p=0.02 and p=0.3 respectively. Conclusion: We found that an increase in serum TSH was positively correlated with components of metabolic syndrome and might be a risk factor for metabolic syndrome in Saudis. Further investigations are essential to further confirm the relationship between TSH and components of metabolic syndrome in Saudis as well as the underlying mechanism(s).

Intercalation of shRNA-plasmid in Mg–Al layered double hydroxide nanoparticles and its cellular internalization for possible treatment of neurodegenerative diseases

In the present work, nanoconjugates of shRNA-plasmid and a non-viral nanoceramic vector, e.g., Mg–Al layered double hydroxide (Mg-Al LDH), were synthesized and intercalated. Subsequently, these particles with an average size of 40–60 nm, were transfected into mammalian neuroblastoma cells (SH-SY5Y). The as prepared Mg–Al LDH was able to protect the incorporated shRNA-plasmid against a range of pH values, DNaseI, endonucleases, and serum components. To test the applicability of the nanoconjugate for future in-vivo studies, serum from three different model experimental animals viz, mouse, rat and guinea pig was used for the serum protection study. Additionally, we showed that prolonged storage at different temperatures does not affect the quality of the nanoconjugate. Using this nanoconjugate to transform cells, a maximum internalization of ∼26% at 24h was achieved. Lastly, we demonstrated effective and safe delivery of the plasmid by measuring GFP production and shRNA-induced knockdown of TNF alpha.

Regulation of Plasmodium sporozoite motility by formulation components

BackgroundThe protective efficacy of the most promising malaria whole-parasite based vaccine candidates critically depends on the parasite’s potential to migrate in the human host. Key components of the parasite motility machinery (e.g. adhesive proteins, actin/myosin-based motor, geometrical properties) have been identified, however the regulation of this machinery is an unknown process.MethodsIn vitro microscopic live imaging of parasites in different formulations was performed and analysed, with the quantitative analysis software SMOOTIn vitro, their motility; their adherence capacity, movement pattern and velocity during forward locomotion.ResultsSMOOTIn vitro enabled the detailed analysis of the regulation of the motility machinery of Plasmodium berghei in response to specific (macro)molecules in the formulation. Albumin acted as an essential supplement to induce parasite attachment and movement. Glucose, salts and other whole serum components further increased the attachment rate and regulated the velocity of the movement.ConclusionsBased on the findings can be concluded that a complex interplay of albumin, glucose and certain salts and amino acids regulates parasite motility. Insights in parasite motility regulation by supplements in solution potentially provide a way to optimize the whole-parasite malaria vaccine formulation.

Platform for the growth and propogation of HEK293 cells and adenovirus viral vector amplification

Background & Aim Industrialization of viral vector production for gene therapy applications demands consistent processes utilizing robust production platforms and raw materials that can meet quality standards and regulatory compliance. Typical vector production schemes involve propagating seed trains and viral amplification in cultures utilizing serum containing media. Although serum-containing media systems deliver high viral vector yields, the serum component has certain disadvantages such as performance variability, high costs, and potential of introducing adventitious agents. Utilizing serum-free chemically-defined medium for growth and amplification addresses these concerns. Here we describe a highly efficient and economical single-use bioreactor platform for producing gene therapy viral vectors with flexibility of using serum-containing or serum-free media. Methods, Results & Conclusion We evaluated multiple serum-free chemically-defined media formulations for adenovirus vector production in the single-use fixed-bed iCELLis® bioreactor. These media were evaluated for ease of adaptation and growth characteristics with HEK293 cells previously adapted to serum-free growth conditions. Process parameters such as multiplicity of infection (MOI) and duration of infection (DOI) were optimized in a shake flask to maximize vector amplification. The process was then transferred into the iCELLis Nano bioreactor. The HEK293 cells were maintained under adherent conditions and growth was monitored until the cell densities reached approximately 200,000 cells/cm2. Complete medium exchange was performed in iCELLis Nano bioreactor without cell removal, followed by viral infection at MOI 50. Harvest was performed 72 hours post infection. Viral titers were quantitated using an adenovirus in-vitro infectivity assay. Viral productivity was generally consistent between the shake flask and the iCELLis Nano bioreactor system and between serum-containing or serum-free chemically-defined medium. These studies demonstrate the iCELLis bioreactor as a versatile scalable technology, and support its use for production of gene therapy vectors in processes using serum-containing or serum-free media.

Proteome Profiling of Exosomes Purified from a Small Amount of Human Serum: The Problem of Co-Purified Serum Components.

Untargeted proteomics analysis of extracellular vesicles (EVs) isolated from human serum or plasma remains a technical challenge due to the contamination of these vesicles with lipoproteins and other abundant serum components. Here we aimed to test a simple method of EV isolation from a small amount of human serum (<1 mL) using the size-exclusion chromatography (SEC) standalone for the discovery of vesicle-specific proteins by the untargeted LC–MS/MS shotgun approach. We selected the SEC fraction containing vesicles with the size of about 100 nm and enriched with exosome markers CD63 and CD81 (but not CD9 and TSG101) and analyzed it in a parallel to the subsequent SEC fraction enriched in the lipoprotein vesicles. In general, there were 267 proteins identified by LC–MS/MS in exosome-containing fraction (after exclusion of immunoglobulins), yet 94 of them might be considered as serum proteins. Hence, 173 exosome-related proteins were analyzed, including 92 proteins absent in lipoprotein-enriched fraction. In this set of exosome-related proteins, there were 45 species associated with the GO cellular compartment term “extracellular exosome”. Moreover, there were 31 proteins associated with different immune-related functions in this set, which putatively reflected the major role of exosomes released by immune cells present in the blood. We concluded that identified set of proteins included a bona fide exosomes components, yet the coverage of exosome proteome was low due to co-purified high abundant serum proteins. Nevertheless, the approach proposed in current work outperformed other comparable protocols regarding untargeted identification of exosome proteins and could be recommended for pilot exploratory studies when a small amount of a serum/plasma specimen is available.

Effect of Knockout Serum Replacement During Postwarming Recovery Culture on the Development and Quality of Vitrified Parthenogenetic Porcine Blastocysts.

The postwarming recovery culture, as one of the steps in cryopreservation process, is directly correlated with the survival and quality of embryos. Generally, recovery medium includes undefined serum or serum components that may cause the instability of results and other problems. The objective of this study was to evaluate the effect of knockout serum replacement (KSR) as a substitute for serum during recovery culture on the development and quality of vitrified parthenogenetic porcine blastocysts. Fetal bovine serum (FBS) was used as a positive control. The expanded blastocysts on day 5 were vitrified by the Cryotop method, and recovered with 10% (v/v) KSR or 10% (v/v) FBS for 48 hours after warming. Survival and hatching rates of vitrified blastocysts were significantly increased by KSR or FBS supplementation. The vitrified blastocysts recovered in KSR or FBS exhibited significantly decreased percentages of membrane damage and apoptosis, and increased total cells. Addition of KSR or FBS during recovery culture significantly reduced reactive oxygen species levels, and improved mitochondrial activity and adenosine triphosphates content in the vitrified blastocysts. Vitrification did not affect the gene expression of PCNA, CDX2, and CPT1, but significantly increased mRNA levels of POU5F1 and uPA. KSR added to the recovery medium significantly upregulated mRNA levels of PCNA and CPT1, and downregulated POU5F1 mRNA levels. The expression levels of PCNA, CDX2, CPT1, and uPA in vitrified blastocysts were significantly upregulated by addition of FBS to recovery medium. Moreover, the BAX: BCL2L1 ratio was similar between fresh and vitrified blastocysts, and KSR or FBS supplementation had no effect on the value. In conclusion, our data showed that KSR supplementation during recovery culture can improve the development and quality of vitrified parthenogenetic porcine blastocysts. These findings provide a useful reference that KSR could be used to replace FBS as a defined serum supplement for recovery culture of vitrified blastocysts.

Discovery of quality-marker ingredients of Panax quinquefolius driven by high-throughput chinmedomics approach

BackgroundQuality control of traditional Chinese medicine (TCM) has always been a hot issue to TCM. However, due to the complexity of TCM ingredients, the current quality standards of TCM have problems that are difficult to guarantee clinical efficacy. American ginseng, the dried roots of Pawajc quinquefolium L. (Araliaceae), is a valuable herbal medicine due to various pharmacological effects and huge health benefit, which are associated with numerous active ingredients such as ginsenosides. Although a large number of studies have investigated the active ingredients of American ginseng, Q-markers reflecting comprehensive review on its efficacies has yet been unrevealed. PurposeThe study aims to discover the Q-markers of Panax quinquefolius (American ginseng), provides a powerful method to clarify the significant ingredents of TCM and help further discovering extensive quality evaluation model,contributing to a significant improvement of TCM quality standard. MethodsMice general status, biochemical indexes assay, urine metabolic profile, and serum metabolic profile were utilized for model replication and efficacy evaluation. The in vitro and in vivo constituents of American ginseng using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS) with Serum Pharmacochemistry of TCM were in-depth investigated. Q-markers that were associated with core markers of therapeutic effects were excavated by a plotting of correlation between marker metabolites and serum constituents (PCMS) approach. ResultsCorrelation analysis of 41 blood and urine labeled metabolites with 14 serum components showed that 24-methyl-7-cholesten-3β-ol, zizybeoside II, betulin, ginsenoside Rd, cinnamyl alcohol, pseudoginsenoside F11 is highly correlated with the therapeutic effects of Compound Zaofan Pill (CZP), while pseudoginsenoside F11 and ginsenoside Rd are highly correlated with the therapeutic effects of American ginseng. The six absorbed blood compounds can be considered as potential Q-markers for compound, of which two compounds, such as pseudoginsenoside F11 and ginsenoside Rd, can be considered as potential Q-markers for American ginseng. ConclusionThe study has demonstrated that the Chinmedomics is an effective, comprehensive and fire-new method for discovering the Q-markers of TCM, and it may be more reasonable choices to establish quality standards of TCM.

Adrenocorticotropin/cortisol ratio – a marker of psoriasis severity

IntroductionThe hypothalamic–pituitary–adrenal (HPA) axis plays a crucial role in systemic homeostasis and hormonal regulation of metabolic and immune functions. A similar HPA axis analog exists in the skin, where it regulates inflammation, cell proliferation and differentiation. Data regarding central HPA axis dysregulation in psoriasis are interesting but so far inconclusive.AimIn the study we attempted to determine whether central HPA axis serum components correlate with psoriasis severity.Material and methodsForty-two patients (10 women and 32 men) hospitalized at the Department of Dermatology participated in the study. None of our patients received any systemic treatment. Venous blood samples were collected at 6.00 AM. The relationship between quantitative variables and psoriasis severity based on the Psoriasis Area and Severity Index (PASI) was assessed with proc logistic in SAS 9.4.ResultsThe effect of adrenocorticotropin/cortisol ratio on the PASI group was OR 3.621 (95% confidence limits 1.217–10.775) for a 0.1 change in ratio (p = 0.02), meaning ACTH/cortisol ratio positively correlates with psoriasis severity. The effect of ACTH and cortisol on the PASI group was not statistically significant, with p-values of 0.30 and 0.23 respectively. Other inflammatory markers such as high-sensitivity C-reactive protein, neutrophils level, LDL, and total cholesterol did not show a significant correlation with PASI score.ConclusionsOur results support the role of HPA axis dysfunction in the complex pathogenesis of psoriasis, showing a positive correlation between morning ACTH/cortisol ratio and disease severity. ACTH/cortisol ratio can be regarded as a new biochemical marker of psoriasis severity worth further studies.

Oviduct proteins in fertilization and early embryo development

The oviduct controls the environment in which the gametes are transported and fuse, and in which embryonic development begins. The ultrastructural topography of the ampulla and isthmus is similar, consisting of ciliated and secretory cells, but a different array of proteins is secreted by each segment along with various serum components. Amino acids are selectively secreted by the oviduct; these amino acids probably interact with the gametes or embryo to facilitate the processes of fertilization and development. An oviduct-specific glycoprotein is synthesized by the ampulla of sheep and cattle in response to oestrogen and secreted mainly from day-1 to day 3 of the ovarian cycle. This oestrus-associated glycoprotein (EGP) has a variable molecular mass of 80-97 kDa and a pI value ranging from 4.7 to 5.5. The bovine (b) and ovine (o) EGP genes are 95.5% identical and consist of 1560 base pairs encoding 519 amino acids containing one N-linked and several O-linked glycosylation sites. The terminal glycosides are N-acetylglucosamine and galactose-N-acetylgalactosamine for bEGP, and fucose, galactose and sialic acid residues are also identified for oEGP. EGP binds to zona pellucida and blastomere membranes, but evidence for EGP binding to sperm membranes is equivocal. After in vitro fertilizaton the proportion of sheep oocytes cleaving was increased in the presence of oEGP, but when single-cell embryos were cultured with oEGP, these cleavage rates were reduced. In addition, consistent positive effects of oEGP were observed on blastocyst formation. Elaboration of the mechanism of synthesis of EGP, its action and its role in fertilization and embryo development is important for our understanding of the events of early pregnancy.

Usefulness of apolipoprotein B-depleted serum in cholesterol efflux capacity assays using immobilized liposome-bound gel beads.

Cholesterol efflux capacity (CEC) in atherosclerotic lesions is the main anti-atherosclerotic function of high-density lipoprotein (HDL). In recent studies, apolipoprotein (apo) B-depleted serum (BDS) obtained with the polyethylene glycol (PEG) precipitation method is used as a cholesterol acceptor (CA) substitution for HDL isolated by ultracentrifugation. However, the suitability of BDS as a CA is controversial. In the present study, CEC obtained from BDS (BDS-CEC) was evaluated based on a parameter, defined as whole-CEC, which was calculated by multiplying CEC obtained using fixed amounts of HDL by cholesterol concentration to HDL-cholesterol (HDL-C) levels in the serum. Significant correlation (r = 0.633) was observed between both CECs. To eliminate systematic errors from possible contamination with serum proteins and low-density lipoprotein (LDL) or very-LDL (VLDL) in BDS-CEC, the deviation of each CEC-BDS from the regression equation was compared with serum protein, LDL, and triglyceride (TG) levels. No correlation was observed between the deviation and the levels of each of these serum components, indicating that the deviations do not derive from systematic error. Further, to evaluate the effects of serum protein on the results, we measured BDS-CEC of reconstituted serum samples prepared using combinations of five levels of serum proteins with five levels of HDL-C. No significant change in BDS-CEC was observed in any combination. These results indicate that BDS-CEC reflects not only the function of HDL but also its concentration in serum.

Environmental signals control transmission stage formation in Plasmodium falciparum

Sexual commitment initiates production of the transmission‐competent gametocyte stage in malaria parasites. Chromatin remodeling events at the ap2‐g locus and activation of the encoded transcription factor are the earliest steps known in this differentiation process. Interestingly, the rate of gametocyte formation is not fixed and variation is thought to depend on environmental cues.Using an assay to probe gametocyte formation in vitro, we found that parasites induce sexual commitment in response to depletion of human serum components. Fractionation experiments identified lysophosphatidylcholine (lysoPC) – a major phospholipid component of serum – as the active host factor. Low micromolar concentrations of lysoPC are sufficient to prevent sexual differentiation in P. falciparum cultured under otherwise commitment‐inducing conditions. Metabolic labeling experiments revealed that parasites readily use lysoPC as a substrate for the synthesis of other lipids, including phosphatidylcholine (PC). In the absence of lysoPC, parasites induce a switch in lipid metabolism that is accompanied by the transcriptional up‐regulation of enzymes used for de novo synthesis of PC (Kennedy pathway). RNA‐Seq data revealed that lysoPC‐depletion further induces the expression of both known (i.e. ap2‐g, gdv1) and new markers that define the transcriptional signature of commitment. Altogether, our results provide unprecedented insights on how malaria parasites integrate external stimuli into the decision‐making process of sexual differentiation. We are currently applying this knowledge in translational research, including drug screens that aim at identifying compounds with malaria transmission‐blocking activity.Support or Funding InformationSupported by a Postdoc. Mobility fellowship from the Swiss National Science Foundation (P300PA_160975)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The efficacy of allergen-immunotherapy in patients with pollen allergy in the Central and Western regions of Ukraine considering the specifics of their sensitization

Various sorts of weed plants with allergenic properties are extended on the territory of Ukraine. Weed’s distribution has regional characteristics. Clinically significant weed allergens are found in pollen from wormwood, ragweed, quinoa, plantain, and thistle. The main treatment for hay fever is allergen immunotherapy. The modern development of molecular diagnostics makes it possible to clearly identify the main allergen of weed pollen and, on this basis, offer the patient a personalized choice of allergen immunotherapy with the possibility of predicting its effectiveness. In order to determine the characteristics of sensitization to weeds in patients of the Central and Western regions of Ukraine and to evaluate the effectiveness of sublingual allergen immunotherapy (SLIT), 1.400 patients from 3 to 65 years of age were examined in these regions. Patients were evaluated general laboratory and instrumental studies, skin prick-tests (SPT), level total serum and specific IgE and component studies. A two-year course of immunotherapy was performed using sublingual allergens of wormwood and ragweed. Evaluation of the effectiveness of SLIT was performed using a visual analogue scale (VAS). According to the results of the study, positive SPT for mugwort was detected 1.2 times more often among patients in the western regions compared with the central regions. Regarding sensitization to ragweed, on the contrary, it was 3.2 times more often observed in patients of the central region. Component diagnostic data confirmed the results of SPT. Patients taking SLIT showed significantly lower VAS (P &lt; 0.05) compared with patients receiving symptomatic therapy. The regional characteristics of sensitization to various types of weeds were found. Latent sensitization to ragweed is increasingly found in patients in the western regions of Ukraine. Use in patients with AR and/or BA SLIT with standardized allergens is an effective and safe treatment method that allows you to quickly control symptoms.&#x0D;

Amiloidosi da transtiretina (ATTR): l’altra faccia della medaglia

Transthyretin amyloidosis (ATTR) is becoming an emerging clinical entity, and is currently the most common form of systemic amyloidosis. ATTR consists of two distinct diseases depending on whether the amyloid fibrils derive from the intact molecule of TTR (ATTR wild-type or senile systemic amyloidosis) or from different mutations occurred in the TTR molecule gene (ATTRm). Total-body scintigraphy with diphosphonates has greatly improved the diagnosis in patients with isolated cardiac involvement for both ATTRm and ATTRwt, thus avoiding the need of endomiocardial biopsy in cases without serum and/or urinary monoclonal component (MC). Heart failure alone, or associated with peripheral and autonomic neuropathy, are the main clinical symptoms in these patients. Particular attention must be paid in order to exclude hypertrophic cardiomiopathy or light chain (AL) forms in patients with MC. Today, besides hepatic transplantation, which is almost reserved to patients with early Val30Met neuropathy, many new therapeutic alternatives can be offered to these patients. Tafamidis, a TTR-stabilizer that has recently proved to be effective in cardiac forms of both ATTRm and ATTRwt, is now ready for clinical use. In addition, drugs silencing the TTR messenger RNA should soon be available for treatment. The many therapeutic opportunities available today for ATTR, strenghten even more the need for an early diagnosis of the disease both in ATTRm and ATTRwt.

From the Editor’s desk…: April 2019

From the Editor’s desk…: April 2019

Characterization of a Manganese‐Containing Nanoparticle as an MRI Contrast Agent

We present relaxivity and physicochemical data for a high relaxivity, manganese‐containing, nanoparticle‐based, and tumor selective MRI contrast agent, SN132D. The nanoparticles have a diameter of 5–6 nm, selected to give a compromise between renal excretion and tumor contrast, based on the Enhanced Permeability and Retention effect. The per manganese relaxivity (r1) is 24.8 ± 0.2 mm–1 Mn s–1 at 1.5 T and 37 °C in water, corresponding to per nanoparticle relaxivity of 347 ± 3 mm–1 s–1. The NMRD profiles in both water and serum showed a peak in r1 in the frequency range 20–40 MHz, which is characteristic of slowly moving paramagnetic systems. The r1 decreases when the temperature is increased from 25 °C to 37 °C, indicating fast exchange dynamics of the coordinated water molecules. The exchange time constant of the whole water molecule, τMO, was 6 ns. The r1 in serum is significantly higher than in water, due to an increase in medium viscosity and chelation of 18.9 ± 2.0 % of MnII ions to human serum components, most likely human serum albumin. The r1 is stable in the pH range 3–10. The per Mn relaxivity is among the highest reported for Mn‐containing systems and far higher than those of the clinically used gadolinium‐based contrast agents.

MRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression invivo were made more than 25 years ago. However, widespread appreciation of invitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such invivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient invivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.