Simultaneous Display of Multiple Foreign Peptides in the FliD Capping and FliC Filament Proteins of the Escherichia coli Flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

A Deletion in the α2B‐Adrenergic Receptor Gene and Autonomic Nervous Function in Central Obesity

Objective: We investigated the impact of a three‐amino acid deletion (12Glu9) polymorphism in the α_2B‐adrenergic receptor gene on autonomic nervous function. The short form (Glu⁹/Glu⁹) of the polymorphism has previously been associated with a reduced basal metabolic rate in obese subjects. Because autonomic nervous function participates in the regulation of energy metabolism, there could be a link between this polymorphism and autonomic nervous function. Research Methods and Procedures: Data of a 10‐year follow‐up study with 126 nondiabetic control subjects and 84 type 2 diabetic patients were used to determine the effects of the 12Glu9 polymorphism on autonomic nervous function. A deep breathing test and an orthostatic test were used to investigate parasympathetic and sympathetic autonomic nervous function. In addition, cardiovascular autonomic function was studied using power spectral analysis of heart rate variability. Results: No significant differences were found in the frequency of the 12Glu9 deletion polymorphism between nondiabetic and diabetic subjects. The nondiabetic men with the Glu⁹/Glu⁹ genotype, especially those with abdominal obesity, had significantly lower total and low‐frequency power values in the power spectral analysis when compared with other men. Furthermore, in a longitudinal analysis of 10 years, the decrease in parasympathetic function was greater in nondiabetic men with the Glu⁹/Glu⁹ genotype than in the men with the Glu⁹/Glu¹² or Glu¹²/Glu¹² genotypes. Discussion: The results of the present study suggest that the 12Glu9 polymorphism of the α_2B‐adrenergic receptor gene modulates autonomic nervous function in Finnish nondiabetic men. In the nondiabetic men with the Glu⁹/Glu⁹ genotype, the general autonomic tone is depressed, and vagal activity especially becomes impaired with time. Furthermore, this association is accentuated by central obesity.     

Severe acute respiratory syndrome coronavirus fails to activate cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells

Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-alpha], IFN-beta, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-alpha/beta-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.     

Acidity and lipolysis by group V secreted phospholipase A(2) strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A(2) (sPLA(2)-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA(2)-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased >60-fold and sPLA(2)-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA(2)-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA(2)-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.     

Recombination Enhances HIV-1 Envelope Diversity by Facilitating the Survival of Latent Genomic Fragments in the Plasma Virus Population

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation process including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.     

Equilibrating errors: reliable estimation of information transmission rates in biological systems with spectral analysis-based methods

Shannon’s seminal approach to estimating information capacity is widely used to quantify information processing by biological systems. However, the Shannon information theory, which is based on power spectrum estimation, necessarily contains two sources of error: time delay bias error and random error. These errors are particularly important for systems with relatively large time delay values and for responses of limited duration, as is often the case in experimental work. The window function type and size chosen, as well as the values of inherent delays cause changes in both the delay bias and random errors, with possibly strong effect on the estimates of system properties. Here, we investigated the properties of these errors using white-noise simulations and analysis of experimental photoreceptor responses to naturalistic and white-noise light contrasts. Photoreceptors were used from several insect species, each characterized by different visual performance, behavior, and ecology. We show that the effect of random error on the spectral estimates of photoreceptor performance (gain, coherence, signal-to-noise ratio, Shannon information rate) is opposite to that of the time delay bias error: the former overestimates information rate, while the latter underestimates it. We propose a new algorithm for reducing the impact of time delay bias error and random error, based on discovering, and then using that size of window, at which the absolute values of these errors are equal and opposite, thus cancelling each other, allowing minimally biased measurement of neural coding.     

Growth performance and feed conversion of Ruspolia differens on plant-based by-product diets

The use of food industry by-products in insect feeds has gained increasing attention recently. However, the understanding of how well the economically valuable edible insect Ruspolia differens (Serville) (Orthoptera: Tettigoniidae) can grow and develop with plant-based by-product feeds is currently lacking. It is important to determine the nutritional requirements, especially protein demand, of this species before developing artificial feeds for mass-rearing. We reared R. differens with four control diets and 12 plant-based by-product diets in which the major protein source came from food industry by-products, including potato-protein, barley mash, barley feed, turnip rape, a mix of broad bean and pea, and a mix of potato, carrot, and apple. We asked whether the performance (development time, survival, and weight), feed conversion, and fatty acid composition and content differed among diet treatments. Furthermore, the 12 experimental by-product diets were designed to reach six protein levels. We found that R. differens can be reared with various by-product diets, but development time, survival, and weight differed among diets. Barley feed, barley mash, and potato protein diets seem to be good options for rearing, and potato glycoalkaloids do not affect the performance of R. differens. Individuals fed on the various by-product diets also differed in their fatty acid composition and content. Increasing protein levels in diet up to 17% enhanced growth, development time, and survival, but no further enhancements were seen when fed diets with protein levels higher than this. The high protein levels decreased feed conversion rate. Our results can be valuable for designing feeds for insect mass-rearing technology. The use of food industry by-products in the diets for R. differens could increase the re-use of local resources and enhance circular economy.     

Modified Lipoprotein-Derived Lipid Particles Accumulate in Human Stenotic Aortic Valves

In aortic stenosis plasma lipoprotein-derived lipids accumulate in aortic valves. Here, we first compared the lipid compositions of stenotic aortic valves and atherosclerotic plaque cores. Both pathological tissues were found to be enriched in cholesteryl linoleate, a marker of extracellularly accumulated lipoproteins. In addition, a large proportion of the phospholipids were found to contain arachidonic acid, the common precursor of a number of proinflammatory lipid mediators. Next, we isolated and characterized extracellular lipid particles from human stenotic and non-stenotic control valves, and compared them to plasma lipoproteins from the same subjects. The extracellular valvular lipid particles were isolated from 15 stenotic and 14 non-stenotic aortic valves. Significantly more apoB-100-containing lipid particles were found in the stenotic than in the non-stenotic valves. The majority of the lipid particles isolated from the non-stenotic valves had sizes (23±6.2 nm in diameter) similar to those of plasma low density lipoprotein (LDL) (22±1.5 nm), while the lipid particles from stenotic valves were not of uniform size, their sizes ranging from 18 to more than 500 nm. The lipid particles showed signs of oxidative modifications, and when compared to isolated plasma LDL particles, the lipid particles isolated from the stenotic valves had a higher sphingomyelin/phosphatidylcholine –ratio, and also higher contents of lysophosphatidylcholine and unesterified cholesterol. The findings of the present study reveal, for the first time, that in stenotic human aortic valves, infiltrated plasma lipoproteins have undergone oxidative and lipolytic modifications, and become fused and aggregated. The generated large lipid particles may contribute to the pathogenesis of human aortic stenosis.     

Unpacking the link between family socioeconomic status and civic engagement during the transition to adulthood: Do work values play a role?

We investigated whether the link between family-of-origin socioeconomic status (SES) and civic engagement in young adulthood is mediated by youth’s work values, that is, the desired characteristics of their current or future jobs. We used data from a Finnish study: 2004 (age 16–18, N_T1?=?1,301); 2011 (age 23–25, N_T2?=?1,096); and 2014 (age 25–27, N_T3?=?1,138). A higher family SES in 2004 predicted youth’s higher civic engagement in 2014. A higher family SES also predicted a lower importance of extrinsic job rewards (e.g., good pay) in 2011, but it was unrelated to the importance of intrinsic job rewards (e.g., learning opportunities). Extrinsic work values, in turn, predicted lower civic engagement in 2014, above and beyond sociodemographic and personality characteristics. Intrinsic work values predicted higher civic engagement. Thus, extrinsic, but not intrinsic, work values partly mediated the link between family SES and youth civic engagement.     

Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin-lipoprotein interactions

Atherosclerosis is initiated when lipoproteins bind to proteoglycans (PGs) in arterial walls. The binding is mediated by apolipoprotein apoB-100 and/or apoE, both of which have binding affinity toward heparin. We developed covalently bound heparin coatings for APTES-modified silica capillaries and SiO(2) chips and carried out capillary electrochromatography (CEC) and quartz crystal microbalance (QCM) studies on the interactions of heparin with selected peptide fragments of apoB-100 and apoE and, for CEC, also with low- and high-density lipoproteins (LDL and HDL), the latter with and without apoE. The peptides are known to mediate interactions of HDL and LDL with arterial PGs. Interactions and affinities were expressed in CEC as retention factors and reduced mobilities and in continuous flow QCM techniques as affinity constants. Both techniques showed heparin interactions to be stronger with apoB-100 peptide than with apoE peptide fragment, and they confirmed that the sulfate groups in heparin play an especially important role in interactions with apoB-100 peptide fragments. In addition, CEC confirmed the importance of sulfate groups of heparin in interactions between heparin and LDL and between heparin and apoE-containing HDL. CEC and QCM acted as excellent platforms to mimic these biologically important interactions, with small sample and reagent consumption.