Surface activity and film formation from the surface associated material of artificial surfactant preparations

AbstractSurfactant proteins B and C (SP-B and SP-C) are present in natural derived surfactant preparations used for treatment of respiratory distress syndrome. Herein the surface activity of an SP-C analogue (SP-C(LKS)), a hybrid peptide between SP-C and bacteriorhodopsin (SP-C/BR) and a model peptide (KL4) was studied with a captive bubble surfactometer (CBS). The peptides were mixed with either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/phosphatidylglycerol (PG) (7:3, by weight) or DPPC/PG/palmitic acid (68:22:9, by weight) at a concentration of 1 mg/ml in HEPES buffer, pH 6.9 and a polypeptide/lipid weight ratio of 0.02–0.03. In some lipid/peptide preparations also 2% of SP-B was included. Adsorption, monitored as surface tension vs. time for 10 min after bubble formation did not show discernible differences for the whole set of preparations. Equilibrium surface tensions of approximately 25 mN/m were reached after 5–10 min for all preparations, although those with SP-C/BR appeared not to reach end point of adsorption within 10 min. Area compression needed to reach minimum surface tension of 0.5–2.0 mN/m was least for the KL4 preparation, about 13% in the first cycle. 3% SP-C(LKS) in DPPC:PG (7:3, by weight) reached minimum surface tension upon 27% compression in the first cycle. If DPPC:PG:PA (68:22:9, by weight) was used instead only 16% area compression was needed and 14% if also 2% SP-B was included. 3% SP-C(LKS) in DPPC:PG (7:3, by weight)+2% SP-B needed 34% compression to reach minimum surface tension. The replenishment of material from a surface associated surfactant reservoir was estimated with subphase depletion experiments. With the 2% KL4 preparation incorporation of excess material took place at a surface tension of 25–35 mN/m during stepwise bubble expansion and excess material equivalent to 4.3 monolayers was found. When 2% SP-B was added to 3% SP-C(LKS) in DPPC:PG (7:3, by weight) the number of excess monolayers increased from 1.5 to 3.6 and the incorporation took place at 30–40 mN/m. When SP-B was added to 3% SP-C(LKS) in DPPC:PG:PA (68:22:9, by weight) the number of excess monolayers increased from 0.5 to 3.4 and incorporation took place at 40–50 mN/m. With 2% SP-C/BR incorporation took place at 40–45 mN/m, frequent instability clicks were observed and excess material of approximately 1.1 monolayer was estimated

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting.publishedVersio

Effects of DHA- Rich n-3 Fatty Acid Supplementation on Gene Expression in Blood Mononuclear Leukocytes: The OmegAD Study

Background: Dietary fish oil, rich in n-3 fatty acids (n-3 FAs), e. g. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), regulate inflammatory reactions by various mechanisms, e. g. gene activation. However, the effects of long-term treatment with DHA and EPA in humans, using genome wide techniques, are poorly described. Hence, our aim was to determine the effects of 6 mo of dietary supplementation with an n-3 FA preparation rich in DHA on global gene expression in peripheral blood mononuclear cells. Methods and Findings: In the present study, blood samples were obtained from a subgroup of 16 patients originating from the randomized double-blind, placebo-controlled OmegAD study, where 174 Alzheimer disease (AD) patients received daily either 1.7 g of DHA and 0.6 g EPA or placebo for 6 months. In blood samples obtained from 11 patients receiving n-3 FA and five placebo, expressions of approximately 8000 genes were assessed by gene array. Significant changes were confirmed by real-time PCR. At 6 months, the n-3 FAs group displayed significant rises of DHA and EPA plasma concentrations, as well as up-and down-regulation of nine and ten genes, respectively, was noticed. Many of these genes are involved in inflammation regulation and neurodegeneration, e. g. CD63, MAN2A1, CASP4, LOC399491, NAIP, and SORL1 and in ubiqutination processes, e. g. ANAPC5 and UBE2V1. Down-regulations of ANAPC5 and RHOB correlated to increases of plasma DHA and EPA levels. Conclusions: We suggest that 6 months of dietary n-3 FA supplementatio

Multi-Organ Expression Profiling Uncovers a Gene Module in Coronary Artery Disease Involving Transendothelial Migration of Leukocytes and LIM Domain Binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) Study

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research