scholarly journals Biomolecular Perturbations in In-Cell Dynamic Nuclear Polarization Experiments

2021 ◽  
Vol 8 ◽  
Author(s):  
Sarah A. Overall ◽  
Alexander B. Barnes
Keyword(s):  
Structural Integrity ◽  
Membrane Integrity ◽  
Embryonic Cell ◽  
Nmr Studies ◽  
Intact Cells ◽  
Biomolecular Structure ◽  
Glass Forming ◽  
The Rich ◽  
And Function ◽  
Cryogenic Conditions

In-cell DNP is a growing application of NMR to the study of biomolecular structure and function within intact cells. An important unresolved question for in-cell DNP spectroscopy is the integrity of cellular samples under the cryogenic conditions of DNP. Despite the rich literature around cryopreservation of cells in the fields of stem cell/embryonic cell therapeutics, cell line preservation and in cryo-EM applications, the effect of cryopreservation procedures on DNP parameters is unclear. In this report we investigate cell survival and apoptosis in the presence of cryopreserving agents and DNP radicals. We also assess the effects of these reagents on cellular enhancements. We show that the DNP radical AMUPol has no effect on membrane permeability and does not induce apoptosis. Furthermore, the standard aqueous glass forming reagent, comprised of 60/30/10 d8-glycerol/D2O/H2O (DNP juice), rapidly dehydrates cells and induces apoptosis prior to freezing, reducing structural integrity of the sample prior to DNP analysis. Preservation with d6-DMSO at 10% v/v provided similar DNP enhancements per √unit time compared to glycerol preservation with superior maintenance of cell size and membrane integrity prior to freezing. DMSO preservation also greatly enhanced post-thaw survival of cells slow-frozen at 1°C/min. We therefore demonstrate that in-cell DNP-NMR studies should be done with d6-DMSO as cryoprotectant and raise important considerations for the progression of in-cell DNP-NMR towards the goal of high quality structural studies.

scholarly journals The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role?

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 54
Author(s):  
Eva Tvrdá ◽  
Miroslava Kačániová ◽  
Andrej Baláži ◽  
Jaromír Vašíček ◽  
Jakub Vozaf ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Semen Quality ◽  
Pearson Correlation ◽  
Interleukin 1 ◽  
Bacterial Load ◽  
Membrane Integrity ◽  
Ros Generation ◽  
Male Gametes ◽  
And Function ◽  
The Impact

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen (n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

scholarly journals Codon harmonization reduces amino acid misincorporation in bacterially expressed P. falciparum proteins and improves their immunogenicity

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Neeraja Punde ◽  
Jennifer Kooken ◽  
Dagmar Leary ◽  
Patricia M. Legler ◽  
Evelina Angov
Keyword(s):  
Protein Structure ◽  
Amino Acid ◽  
Codon Usage ◽  
Dna Sequences ◽  
Structural Integrity ◽  
Host Cells ◽  
Loss Of Function ◽  
Species Specific ◽  
And Function ◽  
The Impact

Abstract Codon usage frequency influences protein structure and function. The frequency with which codons are used potentially impacts primary, secondary and tertiary protein structure. Poor expression, loss of function, insolubility, or truncation can result from species-specific differences in codon usage. “Codon harmonization” more closely aligns native codon usage frequencies with those of the expression host particularly within putative inter-domain segments where slower rates of translation may play a role in protein folding. Heterologous expression of Plasmodium falciparum genes in Escherichia coli has been a challenge due to their AT-rich codon bias and the highly repetitive DNA sequences. Here, codon harmonization was applied to the malarial antigen, CelTOS (Cell-traversal protein for ookinetes and sporozoites). CelTOS is a highly conserved P. falciparum protein involved in cellular traversal through mosquito and vertebrate host cells. It reversibly refolds after thermal denaturation making it a desirable malarial vaccine candidate. Protein expressed in E. coli from a codon harmonized sequence of P. falciparum CelTOS (CH-PfCelTOS) was compared with protein expressed from the native codon sequence (N-PfCelTOS) to assess the impact of codon usage on protein expression levels, solubility, yield, stability, structural integrity, recognition with CelTOS-specific mAbs and immunogenicity in mice. While the translated proteins were expected to be identical, the translated products produced from the codon-harmonized sequence differed in helical content and showed a smaller distribution of polypeptides in mass spectra indicating lower heterogeneity of the codon harmonized version and fewer amino acid misincorporations. Substitutions of hydrophobic-to-hydrophobic amino acid were observed more commonly than any other. CH-PfCelTOS induced significantly higher antibody levels compared with N-PfCelTOS; however, no significant differences in either IFN-γ or IL-4 cellular responses were detected between the two antigens.

scholarly journals Mitochondrial Processes during Early Development of Dictyostelium discoideum: From Bioenergetic to Proteomic Studies

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 638
Author(s):  
Monika Mazur ◽  
Daria Wojciechowska ◽  
Ewa Sitkiewicz ◽  
Agata Malinowska ◽  
Bianka Świderska ◽  
...  
Keyword(s):  
Life Cycle ◽  
Developmental Stages ◽  
Coupling Efficiency ◽  
Slime Mold ◽  
Intact Cells ◽  
Life Cycle Stages ◽  
Early Developmental Stages ◽  
Proteomic Study ◽  
And Function ◽  
Early Developmental

The slime mold Dictyostelium discoideum’s life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria’s structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

scholarly journals Plasma membrane integrity in health and disease: significance and therapeutic potential

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted
Keyword(s):  
Plasma Membrane ◽  
Therapeutic Potential ◽  
Calcium Influx ◽  
Membrane Integrity ◽  
Cell Types ◽  
Physical Parameters ◽  
Membrane Repair ◽  
Plasma Membrane Integrity ◽  
Repair Mechanisms ◽  
And Function

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.

scholarly journals Clinical Relevance of Elastin in the Structure and Function of Skin

2021 ◽  
Author(s):  
Leslie Baumann ◽  
Eric F Bernstein ◽  
Anthony S Weiss ◽  
Damien Bates ◽  
Shannon Humphrey ◽  
...  
Keyword(s):  
Wound Healing ◽  
Clinical Relevance ◽  
Structural Integrity ◽  
Elastic Fiber ◽  
Subcutaneous Fat ◽  
Structure And Function ◽  
Fiber Formation ◽  
Elastic Fibers ◽  
Superficial Dermis ◽  
And Function

Abstract Elastin is the main component of elastic fibers, which provide stretch, recoil, and elasticity to the skin. Normal levels of elastic fiber production, organization, and integration with other cutaneous extracellular matrix proteins, proteoglycans, and glycosaminoglycans are integral to maintaining healthy skin structure, function, and youthful appearance. Although elastin has very low turnover, its production decreases after individuals reach maturity and it is susceptible to damage from many factors. With advancing age and exposure to environmental insults, elastic fibers degrade. This degradation contributes to the loss of the skin’s structural integrity; combined with subcutaneous fat loss, this results in looser, sagging skin, causing undesirable changes in appearance. The most dramatic changes occur in chronically sun-exposed skin, which displays sharply altered amounts and arrangements of cutaneous elastic fibers, decreased fine elastic fibers in the superficial dermis connecting to the epidermis, and replacement of the normal collagen-rich superficial dermis with abnormal clumps of solar elastosis material. Disruption of elastic fiber networks also leads to undesirable characteristics in wound healing, and the worsening structure and appearance of scars and stretch marks. Identifying ways to replenish elastin and elastic fibers should improve the skin’s appearance, texture, resiliency, and wound-healing capabilities. However, few therapies are capable of repairing elastic fibers or substantially reorganizing the elastin/microfibril network. This review describes the clinical relevance of elastin in the context of the structure and function of healthy and aging skin, wound healing, and scars and introduces new approaches being developed to target elastin production and elastic fiber formation.

scholarly journals The Potential Applications of Peroxisome Proliferator-Activated ReceptorδLigands in Assisted Reproductive Technology

PPAR Research ◽  
2008 ◽  
Vol 2008 ◽  
pp. 1-6 ◽  
Author(s):  
Jaou-Chen Huang
Keyword(s):  
Assisted Reproductive Technology ◽  
Reproductive Function ◽  
Reproductive Technology ◽  
Embryonic Cell ◽  
Preimplantation Embryos ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Potential Applications ◽  
And Function

Peroxisome proliferator-activated receptorδ(PPARδ, also known as PPARβ) has ubiquitous distribution and extensive biological functions. The reproductive function of PPARδwas first revealed in the uterus at the implantation site. Since then, PPARδand its ligand have been discovered in all reproductive tissues, including the gametes and the preimplantation embryos. PPARδin preimplantation embryos is normally activated by oviduct-derived PPARδligand. PPARδactivation is associated with an increase in embryonic cell proliferation and a decrease in programmed cell death (apoptosis). On the other hand, the role of PPARδand its ligand in gamete formation and function is less well understood. This review will summarize the reproductive functions of PPARδand project its potential applications in assisted reproductive technology.

scholarly journals Combined Supplementation of Choline and Docosahexaenoic Acid during Pregnancy Enhances Neurodevelopment of Fetal Hippocampus

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Huban Thomas Rajarethnem ◽  
Kumar Megur Ramakrishna Bhat ◽  
Malsawmzuali Jc ◽  
Siva Kumar Gopalkrishnan ◽  
Ramesh Babu Mugundhu Gopalram ◽  
...  
Keyword(s):  
Docosahexaenoic Acid ◽  
Structural Integrity ◽  
Fetal Brain ◽  
Normal Pregnancy ◽  
Saline Control ◽  
Fetal Brain Development ◽  
Essential Nutrient ◽  
Combined Supplementation ◽  
And Function ◽  
The Brain

Choline is an essential nutrient for humans which plays an important role in structural integrity and signaling functions. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, highly enriched in cell membranes of the brain. Dietary intake of choline or DHA alone by pregnant mothers directly affects fetal brain development and function. But no studies show the efficacy of combined supplementation of choline and DHA on fetal neurodevelopment. The aim of the present study was to analyze fetal neurodevelopment on combined supplementation of pregnant dams with choline and DHA. Pregnant dams were divided into five groups: normal control [NC], saline control [SC], choline [C], DHA, and C + DHA. Saline, choline, and DHA were given as supplements to appropriate groups of dams. NC dams were undisturbed during entire gestation. On postnatal day (PND) 40, brains were processed for Cresyl staining. Pups from choline or DHA supplemented group showed significant (p<0.05) increase in number of neurons in hippocampus when compared to the same in NC and SC groups. Moreover, pups from C + DHA supplemented group showed significantly higher number of neurons (p<0.001) in hippocampus when compared to the same in NC and SC groups. Thus combined supplementation of choline and DHA during normal pregnancy enhances fetal hippocampal neurodevelopment better than supplementation of choline or DHA alone.

Antimicrobial mode of action of secretions from the metapleural gland ofMytmecia gulosa(Australian bull ant)

1995 ◽  
Vol 41 (2) ◽  
pp. 136-144 ◽  
Author(s):  
J. A. Mackintosh ◽  
J. E. Trimble ◽  
A. J. Beattie ◽  
D. A. Veal ◽  
M. K. Jones ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Antimicrobial Agents ◽  
Structural Integrity ◽  
Phospholipid Bilayer ◽  
Active Components ◽  
Cytoplasmic Matrix ◽  
Metapleural Glands ◽  
Total Leakage ◽  
Membrane Bound ◽  
And Function

Secretions from exocrine metapleural glands of Myrmecia gulosa (Australian bull ant) exhibit broad-spectrum antimicrobial activity. Treatment of the yeast Candida albicans with metapleural secretion resulted in the rapid and total leakage of K+ions from cells within 10 min. Ultrastructural analysis of the bacteria Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa, and cells and protoplasts of Candida albicans demonstrated gross damage of the cell membrane and aggregation of the cytoplasmic matrix of treated cells. Degradation of membrane-bound organelles was also observed in Candida albicans. The antimicrobially active components of metapleural secretions were nonpolar and interacted with the phospholipid bilayer, causing damage to the structural integrity of liposomes and the release of carboxyfluorescein. The data suggest that the antimicrobial agents in metapleural secretion act primarily by disrupting the structure and function of the phospholipid bilayer of the cytoplasmic membrane.Key words: ant metapleural secretion, antimicrobial, Candida albicans, cytoplasmic membrane.

scholarly journals Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila

Neuron ◽  
2006 ◽  
Vol 51 (2) ◽  
pp. 275 ◽  
Author(s):  
Dhananjay A. Wagh ◽  
Tobias M. Rasse ◽  
Esther Asan ◽  
Alois Hofbauer ◽  
Isabell Schwenkert ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Active Zones ◽  
And Function

Abstract 126: Peroxisomal Proliferator Activated Receptor Alpha Overexpression in Hypertrophied Cardiomyocytes Restores Mitochondrial Integrity and Function

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Sagartirtha Sarkar ◽  
Santanu Rana
Keyword(s):  
Energy Demand ◽  
Cardiac Tissue ◽  
Structural Integrity ◽  
Heart Function ◽  
Ros Generation ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Atp Production ◽  
Receptor Alpha ◽  
And Function

Cardiac tissue engineering is an interdisciplinary field that engineers modulation of viable molecular milieu to restore, maintain or improve heart function. Myocardial workload (energy demand) and energy substrate availability (supply) are in continual flux to maintain specialized cellular processes, yet the heart has a limited capacity for substrate storage and utilization during pathophysiological conditions. Damage to heart muscle, acute or chronic, leads to dysregulation of cardiac metabolic processes associated with gradual but progressive decline in mitochondrial respiratory pathways resulting in diminished ATP production. The Peroxisome Proliferator Activated Receptor Alpha ( PPARα ) is known to regulate fatty acid to glucose metabolic balance as well as mitochondrial structural integrity. In this study, a non-canonical pathway of PPARα was analyzed by cardiomyocyte targeted PPARα overexpression during cardiac hypertrophy that showed significant downregulation in p53 acetylation as well as GSK3β activation levels. Targeted PPARα overexpression during hypertrophy resulted in restoration of mitochondrial structure and function along with significantly improved mitochondrial ROS generation and membrane potential. This is the first report of myocyte targeted PPARα overexpression in hypertrophied myocardium that results in an engineered heart with significantly improved function with increased muscle mitochondrial endurance and reduced mitochondrial apoptotic load, thus conferring a greater resistance to pathological stimuli within cardiac microenvironment.

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