Differential regulation and properties of angiopoietin-like proteins 3 and 4

Angiopoietin-like protein 3 and 4 (Angptl3 and Angptl4) are two members of the angiopoietin-like family of proteins. These two closely related proteins have been reported to similarly affect lipid metabolism through their capacity to inhibit lipoprotein lipase. We undertook a series of studies to compare the structure, function, and regulation of Angptl3 and Angptl4. Previously, we reported that Angptl4 exists as variable-sized oligomers that contain intermolecular disulfide bonds. We now have evidence that although there are no intermolecular disulfide bonds evident in Angptl3, higher molecular weight forms do exist. In addition, Angptl4 exhibits a widespread distribution of tissue expression, while Angptl3 is exclusively expressed in the liver. Treatments with various ligands of nuclear receptors reveal that Angptl3 is a target gene of liver X receptor, while Angptl4 expression is activated by ligands of all peroxisome proliferator-activated receptors. Expression of Angptl4 in adipose tissue and liver is induced by fasting, while Angptl3 expression is not appreciably affected by nutritional status. We suggest that the differential regulation of Angptl3 and Angptl4 by sites of expression, nutritional status, and ligands of nuclear receptors may confer unique roles of each in lipoprotein metabolism.     

The role of Fas in the immune system biology of IL-2R alpha knockout mice: interplay among regulatory T cells, inflammation, hemopoiesis, and apoptosis

Introducing lpr mutation prevents early mortality associated with IL-2Ralpha knockout (KO) mice, prompting us to determine the role of Fas in the immune system biology of IL-2Ralpha KO mice. Consistent with a defect in CD4+CD25+ regulatory T (Treg) cell expression, spontaneous lymphocyte activation in lymphoid organs was observed in 6-wk-old mice. In 16- to 22-wk-old mice, infiltration of leukocytes was observed in bone marrow, colon, lung, pancreas, lacrimal gland, and salivary gland, but not in heart, thyroid, liver, stomach, small intestine, ovary, and kidney. In the lymphocytes-infiltrated bone marrow, B cell lymphopoiesis was blocked at pro-B to pre-B/immature B stage, culminating in an age-dependent B cell loss in the periphery. These phenotypes were also observed in IL-2Ralpha KO mice bearing the lpr mutation (DM mice), indicating Treg cell function and the phenotypes attributed directly to Treg cell abnormality are largely Fas-independent. However, anemia and body weight loss were partially prevented, tissue cell apoptosis was inhibited, and lifespan was improved in the DM mice, demonstrating Fas-dependent elements in these processes. Our age-dependent, lifelong analysis of IL-2Ralpha KO and DM mice supports a CD4+CD25+ Treg cell-based mechanism for the abnormal immune system biology observed in IL-2Ralpha KO mice and provides a global view of the interplays among Treg cells, multiorgan inflammation, hemopoiesis, and apoptosis.     

Hybrid-EKF: Hybrid model coupled with extended Kalman filter for real-time monitoring and control of mammalian cell culture

In a decade when Industry 4.0 and quality by design are major technology drivers of biopharma, automated and adaptive process monitoring and control are inevitable requirements and model-based solutions are key enablers in fulfilling these goals. Despite strong advancement in process digitalization, in most cases, the generated datasets are not sufficient for relying on purely data-driven methods, whereas the underlying complex bioprocesses are still not completely understood. In this regard, hybrid models are emerging as a timely pragmatic solution to synergistically combine available process data and mechanistic understanding. In this study, we show a novel application of the hybrid-EKF framework, that is, hybrid models coupled with an extended Kalman filter for real-time monitoring, control, and automated decision-making in mammalian cell culture processing. We show that, in the considered application, the predictive monitoring accuracy of such a framework improves by at least 35% when developed with hybrid models with respect to industrial benchmark tools based on PLS models. In addition, we also highlight the advantages of this approach in industrial applications related to conditional process feeding and process monitoring. With regard to the latter, for an industrial use case, we demonstrate that the application of hybrid-EKF as a soft sensor for titer shows a 50% improvement in prediction accuracy compared with state-of-the-art soft sensor tools.     

Palmitate exacerbates bisphenol A toxicity via induction of ER stress and mitochondrial dysfunction

Combined exposure to dietary nutrients and environmental chemicals may elicit significantly different physiological effects than single exposures. Exposure to dietary saturated fats and environmental toxins is a physiologically-significant dual exposure that is particularly associated with lower socioeconomic status, potentially placing these individuals at heightened risk of xenobiotic toxicities. However, no prior studies have examined interactions between specific lipids and environmental xenobiotics in modulating cellular health. Using primary mouse embryonic fibroblasts, we have discovered that prior exposure to the saturated fatty acid, palmitate, exacerbates cellular toxicity associated with the industrial plasticizer, bisphenol A (BPA). Cell death upon BPA exposure following palmitate pre-treatment was greater than that occurring with either exposure alone. Mechanistically, cell death was preceded by increased endoplasmic reticulum stress and loss of mitochondrial membrane potential in palmitate plus BPA exposed cells, leading to increased caspase-3 cleavage and subsequent apoptosis. Interestingly, inclusion of the unsaturated fatty acid, oleate, along with palmitate during the pre-treatment period completely abrogated the ER stress, mitochondrial toxicity, and cell death induced by subsequent exposure to BPA. Thus, our data identify for the first time an important interaction between a fatty acid and an environmental toxin and have implications for developing nutritional interventions to mitigate the deleterious effects of such xenobiotic exposures.     

Gradients with depth in electrospun fibrous scaffolds for directed cell behavior

A major obstacle in creating viable tissue-engineered constructs using electrospinning is the lack of complete cellularization and vascularization due to the limited porosity in these densely packed fibrous scaffolds. One potential approach to circumvent this issue is the use of various gradients of chemical and biophysical cues to drive the infiltration of cells into these structures. Toward this goal, this study focused on creating durotactic (mechanical) and haptotactic (adhesive) gradients through the thickness of electrospun hyaluronic acid (HA) scaffolds using a unique, yet simple, modification of common electrospinning protocols. Specifically, both mechanical (via cross-linking: ranging from 27-100% modified methacrylated HA, MeHA) and adhesive (via inclusion of the adhesive peptide RGD: 0-3 mM RGD) gradients were each fabricated by mixing two solutions (one ramping up, one ramping down) prior to electrospinning and fiber collection. Gradient formation was verified by fluorescence microscopy, FTIR, atomic force microscopy, and cellular morphology assessment of scaffolds at different points of collection (i.e., with scaffold thickness). To test further the functionality of gradient scaffolds, chick aortic arch explants were cultured on adhesive gradient scaffolds for 7 days, and low RGD-high RGD gradient scaffolds showed significantly greater cell infiltration compared with high RGD-low RGD gradients and uniform high RGD or uniform low RGD control scaffolds. In addition to enhanced infiltration, this approach could be used to fabricate graded tissue structures, such as those that occur at interfaces.     

Bioprocessing in the Digital Age: The Role of Process Models

In this age of technology, the vision of manufacturing industries built of smart factories is not a farfetched future. As a prerequisite for Industry 4.0, industrial sectors are moving towards digitalization and automation. Despite its tremendous growth reaching a sales value of worth $188 billion in 2017, the biopharmaceutical sector distinctly lags in this transition. Currently, the challenges are innovative market disruptions such as personalized medicine as well as increasing commercial pressure for faster and cheaper product manufacturing. Improvements in digitalization and data analytics have been identified as key strategic activities for the next years to face these challenges. Alongside, there is an emphasis by the regulatory authorities on the use of advanced technologies, proclaimed through initiatives such as Quality by Design (QbD) and Process Analytical Technology (PAT). In the manufacturing sector, the biopharmaceutical domain features some of the most complex and least understood processes. Thereby, process models that can transform process data into more valuable information, guide decision‐making, and support the creation of digital and automated technologies are key enablers. This review summarizes the current state of model‐based methods in different bioprocess related applications and presents the corresponding future vision for the biopharmaceutical industry to achieve the goals of Industry 4.0 while meeting the regulatory requirements.     

Identification of bacteria in culture negative and polymerase chain reaction (PCR) positive intraocular specimen from patients with infectious endopthalmitis

A novel Denaturing High-Performance Liquid Chromatography (dHPLC)-based technique allows rapid high-resolution analysis of PCR products. We show the application of this PCR/dHPLC approach for direct detection and identification of bacterium from the Eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endopthalmitis and to differentially identify the culture negative cases and initiate appropriate therapy. The aim of this study is to identify culture negative PCR positive cases by the application of PCR based DNA sequencing. A total of 116 intraocular specimens were subjected for the study. Sixty-nine different bacteria were identified using dHPLC based DNA sequencing of which predominant ones were Gram-positive bacteria and cannot be cultured by conventional methods. Forty eight different bacteria detected in this study is being reported for the first time in infectious endopthalmitis.     

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro–lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.     

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

Background

Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.

Methodology/Principal Findings

We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.

Conclusions/Significance

We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.     

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.