The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.

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Artificial insemination (AI) using cryopreserved sperm has an important positive impact on cattle production. Fertility is the most critical trait controlling livestock production; however, molecular, cellular, and physiological determinants of bull fertility and sperm freezability are not well understood. Better understanding of molecular, cellular, and physiological underpinnings of bull fertility may increase the success rate of AI. The objective of this study was to test the hypothesis that expression dynamics of sperm nuclear proteins, post-translational modifications (PTM) of sperm Histone 4 (H4), and seminal plasma metabolome are associated with bull fertility and sperm freezability (P = 0.043). Flow cytometry experiments were conducted to quantify H4 and acetylated histone 4 (H4ac) in sperm from high and low fertility Holstein bulls. The analysis of flow cytometry experiments clarified that retained levels of H4ac in bull sperm are associated with bull fertility. In addition, gas chromatography-mass spectrometry (GC-MS) was applied to ascertain the amino acid concentration of seminal plasma from bull semen with various freezability. A total of 21 amino acids and isomers were identified, and phenylalanine was positively associated with sperm post-thaw viability (r = 0.57, P-value = 0.043). Lastly, a quantitative western blotting experiment was utilized to ascertain relative quantification of sperm nuclear proteins including protamine 1 (PRM1), protamine 2 (PRM2), Histone 3 (H3), and H4. Also, sperm functional parameters including acrosome reaction, DNA fragmentation index, PAWP expression were analyzed using flow cytometry. In addition, immunocytochemistry experiments were applied to analyze sperm chromatin decondensation ability. The analyses of western blotting experiments revealed that the relative abundance of PRM2 in poor freezability sperm (PF) was greater than those in good freezability sperm (GF) (P = 0.0259). The relative abundance of retained H3 was greater in PF bulls than in GF bulls (1.02 ± 0.005 and 0.969 ± 0.021, respectively; P = 0.0272). There was a positive correlation between the abundance of retained H4 and sperm decondensation state (r = 0.71, P = 0.05). These results are important because they can help advance fundamental andrology and the assisted reproductive technologies both for cattle and other mammals, including humans and endangered species.

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No description available.

Non-alcoholic fatty liver disease (NAFLD) represents the major cause of pediatric chronic liver pathology in the United States. The objective of this study was to investigate the effect of partial substitution of dietary lard by an isocaloric amount of olive or coconut oil on endpoints of NAFLD. Thirty-eight 15-d-old Iberian pigs housed in pens balanced for weight and sex were randomly assigned to receive 1 of 3 hypercaloric high-fructose high-fat (HFF) diets for 10 weeks: 1) lard (LAR; n=5 pens), 2) lard + olive oil (OLI, n=10), and 3) lard + coconut oil (COC; n=10). Additional pigs (BSL, n=4) were fed a eucaloric diet to establish baseline values. Animals were euthanized at 85 d of age after blood sampling. Liver tissue was collected for histology, metabolomics, and transcriptomics. Compared with BSL, OLI decreased high-density lipoproteins, phosphatidylcholines (PC), and total cholesterol in blood, and increased acylcarnitines in liver, whereas COC increased triacylglycerides (TAGs) in liver and blood. All HFF diets increased bile acids in liver, and decreased choline and fibroblast growth factor 19 in liver and blood. OLI and COC increased hepatic steatosis, necrosis, ballooning, and composite lesion score compared with LAR. OLI decreased gene expression of carnitine O-palmitoyltransferase 1, and COC increased expression of fatty acid binding proteins and acyl-CoA synthetase. In conclusion, partial replacement of dietary lard with olive and coconut oil dysregulated acylcarnitine metabolism and lipogenesis in the liver, increasing the severity of NAFLD in juvenile pigs.

Introduction: Gestational diabetes mellitus (GDM) is a metabolic disorder that has been defined as glucose intolerance that is first identified during pregnancy. The etiology of GDM is not yet fully understood, but there are several risk factors that appear to contribute to its development such as advanced age at pregnancy, family history of type 2 diabetes mellitus, and a previous history of GDM. The discovery of predictive GDM biomarkers has the potential to enable early GDM detection and lead to earlier diagnosis and preventative interventions. Objective: Perform metabolomics analysis on plasma samples collected at pre-conception and at 26-weeks gestation to investigate metabolic differences between participants of the gestational diabetes prevention (GDP) clinical trial who developed GDM and those who did not. Methods: Targeted metabolomics, comprised of primary metabolomics, biogenic amines, and lipidomics assays, was performed using UPLC-MS on plasma samples collected from a subset of 30 participants that completed the GDP study at preconception and 26 weeks gestation. The samples used for this analysis were from participants who developed GDM (n=19) and those who did not (n=11) in their pregnancy following their participation in the GDP study. Results: Multivariate analysis revealed indoxyl sulfate as significantly higher in the GDM group at both preconception and at 26 weeks gestation (VIP scores > 2.9). Preconception samples collected at the end of the GDP intervention study PC 38:0 was higher in the GDM group versus the non-GDM group (p < 0.05) whereas thymidine was lower in the GDM group (p < 0.05), in addition to numerous cell membrane lipids (VIP > 2.0). At 26 weeks gestation, D-glucuronic acid was higher in the GDM group versus the non-GDM group (p < 0.03), while LPE 22:6, SM 18:1 (22:4), PE 38:6, PE 40:6, PE 40:7, and PE (O-38:0) were lower in the GDM group (p < 0.04), in addition to numerous cell membrane lipids (VIP > 2.0). Discussion: The differences observed between the GDM and non-GDM groups at the two plasma collection time points may suggest metabolic alterations associated with GDM-induced metabolic dysregulation. These findings may help direct future research to focus on changes in lipid metabolism during pre-pregnancy for possible biomarkers of GDM. Repeat studies with diverse cohorts are needed to help identify a panel of metabolites that may serve as early biomarkers of GDM.

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for rapid metabolic responses. Taken together, our analysis could be used as an initial road map for the identification of different pathways that could be used to augment the rapid response of plants to abiotic stress. In addition, it highlights the important role of glutathione in initial stage of light stress response. Light-induced rapid systemic signaling and systemic acquired acclimation (SAA) are thought to play an important role in the response of plants to different abiotic stresses. Although molecular and metabolic responses to light stress have been extensively studied in local leaves, and to a lesser degree in systemic leaves, very little is known about the metabolic responses that occur in the different tissues that connect the local to the systemic leaves. These could be important in defining the specificity of the systemic response as well as in supporting the propagation of different systemic signals, such as the reactive oxygen species (ROS) wave. Here we report that local application of light stress to one rosette leaf resulted in a metabolic response that encompassed local, systemic and transport tissues (tissues that connect the local and systemic tissues), demonstrating a high degree of physical and metabolic continuity between different tissues throughout the plant. We further show that the response of many of the systemically altered metabolites could be associated with the function of the ROS wave, and that the level of eight different metabolites is altered in a similar way in all tissues tested (local, systemic, and transport tissues). These compounds could define a core metabolic signature for light stress that propagates from the local to the systemic leaves. Taken together, our findings suggest that metabolic changes occurring in cells that connect the local and systemic tissues could play an important role in mediating rapid systemic signaling and systemic acquired acclimation to light stress.

Metabolomics is used to reduce the complexity of plants and to understand the underlying pathways of the plant phenotype. The metabolic profile of plants can be obtained by mass spectrometry or liquid-state NMR. The extraction of metabolites from the sample is necessary for both techniques to obtain the metabolic profile. This extraction step can be eliminated by making use of high-resolution magic angle spinning (HR-MAS) NMR. In this review, an HR-MAS NMR-based workflow is described in more detail, including used pulse sequences in metabolomics. The pre-processing steps of one-dimensional HR-MAS NMR spectra are presented, including spectral alignment, baseline correction, bucketing, normalisation and scaling procedures. We also highlight some of the models which can be used to perform multivariate analysis on the HR-MAS NMR spectra. Finally, applications of HR-MAS NMR in plant metabolomics are described and show that HR-MAS NMR is a powerful tool for plant metabolomics studies.

Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown. In this study, we combined morphological, immunochemical, proteomic, lipidomic, and metabolomic analyses to obtain an integrated understanding of the mechanisms underlying microvesicle generation during the storage of red blood cell concentrates. Our data indicate that changes in membrane organization, triggered by altered protein conformation, constitute the main mechanism of vesiculation, and precede changes in lipid organization. The resulting selective accumulation of membrane components in microvesicles is accompanied by the recruitment of plasma proteins involved in inflammation and coagulation. Our data may serve as a basis for further dissection of the fundamental mechanisms of red blood cell aging and vesiculation, for identifying the cause-effect relationship between blood bank storage and transfusion complications, and for assessing the role of microvesicles in pathologies affecting red blood cells.