bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Multi‐Resistance Bacteria by Acetone Extract of Cola acuminata
Authors: Telles Caroline M , McKinsey Cayla , Gray Wesley
Published: June 2021

The rapid emergence of multidrug resistant pathogens has created an urgent need for discovering novel antibiotics with natural plant products as potential antimicrobial agents. Cola acuminata (Bizzy Nut), possesses a variety of bioactive compounds which exhibit important bioactive activities against the growth of certain mammalian cell lines, bacteria and fungi. However, the specific chemicals responsible for the antimicrobial bioactivity have not been identified. Therefore, we investigated a bioactive extract from Cola acuminata for the presence of antimicrobial activity using two multidrug resistant pathogenic bacterial species, Staphylococcus aureus and Klebsiella pneumoniae. The specific bioactive compounds present in C. acuminata was partially purified using sequential extraction with solvents of increasing polarity, followed by SPE purification. The antimicrobial activity associated with each extraction was evaluated using the agar diffusion and MIC assay screened with Staphylococcus aureus as a model multidrug resistant pathogenic organism. An enriched antimicrobial activity (Biz‐3w) obtained from the acetone extract (Biz‐3) and SPE purification was effective against both Gram‐positive and Gram‐negative bacteria. We observed a dose and time dependent inhibition of Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis with an effective dose (ED50) of 4 ug/ml, 3 ug/ml, and 3.2ug/ml respectively. The order of potency of Biz‐3w was Bacillus subtilis ≥ Staphylococcus aureus > Enterococcus faecalis. The results obtained in this study provide preliminary evidence of the presence of secondary metabolites in C. acuminata capable of inhibiting multidrug resistant bacteria.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐sensing Receptor Internalization is β‐arrestin‐dependent and Modulated by Allosteric Ligands
Authors: Bräuner‐Osborne Hans , Mos Iris , Jacobsen Stine Engesgaard , Foster Simon Richard
Published: June 2021

G protein‐coupled receptor (GPCR) internalization is a complex process responsible for the transport of GPCRs from the cell‐surface into intracellular compartments. Receptor internalization is crucial for the termination of GPCR activity and in some cases linked to the initiation of G protein‐independent signalling pathways and endosomal receptor signalling. Internalization has been widely studied in class A GPCRs, where the most prevalent mechanism after receptor activation is initiated by kinase‐mediated phosphorylation of intracellular loops and the C‐terminus, followed by β‐arrestin recruitment and dynamin driven internalization via clathrin‐coated pits. However, the mechanism and clinical relevance in class B and C GPCRs remain largely unknown.

The extracellular calcium‐sensing receptor (CaSR) is a prototypical class C GPCR that plays a pivotal role in calcium homeostasis due to continuous sensing of small changes in blood ionized calcium (Ca2+) levels. Impressively, CaSR is able to tightly control its activity despite being constantly exposed to its endogenous agonist. Previous studies investigating CaSR internalization were based on functional assays, and microscopy‐ or flow cytometry‐based analyses. However, there is no general agreement on the pathway(s) involved in CaSR internalization. Overall, the mechanism of CaSR remains to be poorly understood.

In this study, we aimed to investigate CaSR internalization using the recently developed TR‐FRET‐based real‐time internalization assay for the first time. We demonstrate that Ca2+ induces CaSR internalization in a concentration‐dependent manner with an EC50 of 3.3 mM, which aligns well with the IP1 response with an EC50 of 3.2 mM. We show that internalization can be positively modulated by NPS R‐568 and negatively modulated by NPS 2143. Through the usage of CRISPR‐Cas9 edited HEK293 cells, we demonstrate that CaSR internalization is β‐arrestin dependent.

Support or Funding Information

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement NO 675228, the Lundbeck Foundation, the Carlsberg Foundation, the Augustinus Foundation and the Toyota Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Osteogenic and chondrogenic differentiation effect of neohesperidin dihydrochalcones in human adipose‐derived stem cells
Authors: Heo Hee Sun , Cha Ji Hyun , Lee Jong Hun
Published: June 2021

Reactive oxygen species (ROS) are highly reactive which cause damage to various cellular functions such as proliferation, differentiation, and survival. Nuclear factor erythroid 2‐related factor‐2 (Nrf2) promotes expression of antioxidant enzyme such as heme oxygenase 1 (HO‐1) and NAD(P)H:Quinone Oxidoreductase 1 (NQO‐1). Neohesperidin is a compound originated from peel of citrus fruits and its hydrogenated form, neohesperidin dihydrochalcone (NHDC) has antioxidant activity. Human adipose‐derived stem cells (hADSC) are multipotent adult stem cells responsible for the regeneration of fat, bone and cartilage. Thus, we observed the antioxidant effects of NHDC derivatives on hADSC through Nrf2 activation. Upregulation of ROS inhibits osteogenic differentiation of hADSCs, in part through inhibition of signaling pathway, which is essential for bone development and maintenance. ROS levels regulate inhibition of proliferation and modulate initiation of the hypertrophic changes in chondrocytes. Cellular viability and compounds' proper concentrations were set by MTS assay. The effects of these compounds on suppressing oxidative stress and hADSCs differentiation into osteocytes and chondrocytes was investigated using Alizarin Red staining, Alcian Blue staining, reverse‐transcriptase polymerase chain reaction (RT‐PCR), and Western blotting. The results demonstrate that NHDC derivatives play an efficacious role in inducing osteogenic and chondrogenic differentiation of hADSCs through reducing cellular oxidative stress.

Support or Funding Information

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education 2018R1D1A1B07049077

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessment of Cellular Respiration and Oxidative Phosphorylation in Cocks Semen after Freezing
Authors: Nikitkina Elena , Musidray Artem , Timofeeva Svetlana , Krutikova Anna , Plemyashov Kirill , Pleshanov Nikolay
Published: June 2021

The biochemical processes associated with the generation of energy (ATP) ‐ oxidative phosphorylation and glycolysis ‐ occupy a central place in the vital activity of cells. Numerous studies have established that the ratio of respiration and phosphorylation is a sensitive indicator of the functional state of the cell and the whole organism. Oxidative phosphorylation (OXPHOS) is the metabolic pathway in which cells use enzymes to oxidize nutrients, thereby releasing energy which is used to produce adenosine triphosphate (ATP). OXPHOS is the terminal process of cellular respiration (CR). Aim of the study was to evaluate the ratio of respiration and phosphorylation in cocks sperm after freezing. Semen from 10 cocks was frozen. CR was assessed by the polarographic method. OXPHOS can be evaluated by the reaction CR on adding 2,4‐dinitrophenol (2,4 ‐ DNP ) to semen sample. 2,4 ‐ DNP disrupt the proton gradient by carrying protons across a membrane and uncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane. As a result it increases respiration rate. If the respiration rate increases – there is good OXPHOS. Results are depicted as ranges, percentages of totals and averages ± SEs. The addition of 2,4 ‐ DNP to sperm samples caused a significant increase in CR (1.8 ± 0.40 times). This suggests that the CR of freshly diluted cocks sperm is highly associated with phosphorylation. We observed a drop in respiratory stimulation by 2,4 ‐ DNP (1,7±0,11 times) after thawing. This is an indication of an increase in free oxidation not associated with oxidative phosphorylation. The CR stimulation by 2,4‐DNP in the frozen samples ranged from 1.4 to 2.3, and the fertility of eggs ranged from 24 to 95%. The correlation between egg fertilization and stimulation of respiration by 2,4‐DNP was high (r = 0,76, P <0,01). Assessment of oxidative phosphorylation can serve as a marker of sperm viability after freezing and as criterion for predicting the fertilizing ability.

Support or Funding Information

Authors acknowledge financial support from Russian Science Foundation, Grant No: 18‐16‐00071

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NAG‐1/GDF15 inhibits HFD‐induced obesity and inflammation by modulating gut microbiota
Authors: Sang Tingting , Guo Chenjie , Guo Dandan , Na Kun , Wu Kaikai , Li Kang , Wang Xingya
Published: June 2021

Nonsteroidal anti‐inflammatory drug‐activated gene (NAG‐1) or growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor beta (TGF‐β) superfamily. Mice expressing NAG‐1/GDF15 have been shown to be resistant to high fat diet (HFD)‐induced obesity and inflammation. However, the exact mechanism underlying the anti‐obesity effects of NAG‐1/GDF15 remains unknown. The objective of this study was to examine whether the anti‐obesity effects of NAG‐1/GDF15 is associated with gut microbiota modulation and gut barrier function regulation. First, we confirmed that NAG‐1/GDF15 Tg mice are resistant to dietary induced obesity and inflammation. These mice also demonstrated improved insulin sensitivity compared to Wt littermates. We then found these phenotypic differences were correlated with differences in the composition of gut microbiota, gut barrier function and short chain fatty acids compositions in feces as determined by standard molecular biological methods. In addition, HFD reduced the expression of Cyp7a1, Cyp7b1, Cyp27a1, Ntcp, Mdr2, and Oatp that regulate bile acid metabolism in liver. However, the expression of these genes were increased in NAG‐1/GDF15 Tg mice. Furthermore, antibacterial peptide (Reg3g, Lyz‐1 and Defa) and MUC2 in small intestine were up‐regulated in NAG‐1/GDF15 Tg mice. We also found that NGA‐1/GDF15 down‐regulate the expression of TLR4 and its downstream signals in fat, liver and small intestines at both mRNA and protein levels as determined by Western blot and qRT‐PCR. Finally, we transplanted feces from NAG‐1/GDF15 mice into pseudo germ‐free mice and fed the mice with HFD and found that a shift in gut microbiota composition from NAG‐1/GDF15 mice could inhibit the development of obesity and inflammation. Taken together, our results indicate that the protection role of NAG‐1/GDF15 against HFD‐induced obesity and inflammation is mediated, at least in part, by the modulation of gut microbiota, function of gut barrier, and bile acid metabolism.

Support or Funding Information

National Natural Science Foundation of China Grant No. 81400822.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Reassessment of Phosphatidylinositol Transfer Protein Alpha's Growth Factor Signaling Role
Authors: McDermott Mark I. , Diz Ramiro , Hur Seong , Lete Marta G. , Applebee Christopher J. , Grabon Aby , Tripathi Ashutosh , Wakelam Michael J. O. , Larijani Banafshe , Bankaitis Vytas A.
Published: June 2021

In mammals, the START‐like phosphatidylinositol transfer proteins (PITPs) remain an understudied group of proteins implicated in human health and disease. Phosphatidylinositol transfer protein alpha (PITPa) is linked to human epidermal growth factor receptor (HER2)‐positive metastatic breast cancer drug resistance, and muscular dystrophy, but its precise mechanism of action is unclear. Current dogma states that PITPa is essential for growth factor signaling through the epidermal growth factor (EGFR) and netrin signaling pathways, but little data has emerged supporting these claims. Data from our laboratory, from multiple experimental systems, strongly dispute PITPa requirement in these pathways. Strikingly, mouse and human cell lines lacking PITPa sense and chemotax towards EGF, and signaling responses downstream of EGFR‐stimulation are unimpaired, including phosphatidylinositol (3,4,5)P3 generation, and phosphorylation of protein kinase B (Akt). Surprisingly, lack of PITPa in human cells results in potentiation of Akt phosphorylation (threonine 308) following EGFR stimulation, indicating that contrary to current thinking, PITPa may actually repress this pathway. The dispensability of PITPa for EGF‐signaling explains the remarkable absence of EGF‐signaling associated phenotypes in PITPa nullizygous (Pitpa0/0) mice. We have previously reported that Pitpa0/0 animals exhibit a complex phenotype including spinocerebellar neurodegeneration characterized by gliosis and neuronal apoptosis of cerebellum, and hindbrain, and that neuronal specific eviction of embryonic PITPa and PITPb results in progeny born lacking a forebrain. Axon guidance and mouse forebrain development require netrin‐1 signaling through the netrin receptor (DCC), a process reported to require PITPa. We demonstrate that while compromise of netrin‐signaling results in structural brain derangements, these defects do not result from absence of PITPa. Furthermore, we fail to reproduce the previously reported physical interaction between the netrin receptor (DCC) and PITPa. In light of our findings the ‘essential’ role of PITPa in growth factor signaling must be reevaluated.

Support or Funding Information

This work was supported by the Robert A. Welch Foundation (BE0017), and a National Institutes of Health grant (GM44530) awarded to Vytas A. Bankaitis

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased hexokinase interactions with mitochondria protect against cell damage
Authors: Sampe David
Published: June 2021

Deaths due to cancer and ischemic heart disease are the most common; dysfunction of mitochondrial energy (ATP) metabolism is key in these pathologies. Therefore, therapeutic strategies, which remain elusive, have focused on energetic metabolism. Cells use glucose and oxygen in glycolysis and oxidative phosphorylation to generate ATP. The cytosolic protein hexokinase (HK) plays a crucial role in glycolysis by catalyzing the breakdown of glucose to produce most of the ATP generated from mitochondria. For efficient glycolysis, HK must access the mitochondrial ATP, which flows out via the voltage dependent anion channel (VDAC), a 30‐kDa protein in the outer mitochondrial membrane (OMM). Cancer cells grow and proliferate by increasing access to ATP via increased HK‐VDAC associations. This strategy has potential implications for protection against cardiac ischemic injury. Ischemia promotes dissociation of HK from VDAC leading to cardiac damage, but cardio‐protective interventions can increase HK‐VDAC association, preserving function. The overall objective is to determine the biophysical and biochemical features that lead to HK‐VDAC association or dissociation. HK, a 100‐KDa dimeric protein, has 918 amino acids in two domains: an N‐terminus and C‐terminus joined by a linker a‐helix. HK interactions with mitochondria are not well known but, HK, with its non‐polar sequence of 15 amino acids on the N‐terminus, may insert in the OMM close to VDAC. Glucose and glucose‐6‐phospate have distinct binding sites on HK. Glucose binds at K173, D209, and E260 in the N‐terminus and at K621, D657, and E708 in the C‐terminus. The Brown Deer High School SMART Team designed a model of HK using 3‐D printing technology to investigate structure‐function relationships. Understanding HK‐VDAC binding mechanisms could provide better targeted approaches to manage pathologies, including cancer, cardiac and neurodegenerative diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioinformatic Identification of Candidate miRNA Biomarkers for Traumatic Brain Injury
Authors: Smothers Christine , O'Connell Grant C
Published: June 2021

The identification of peripheral blood biomarkers which are associated with traumatic brain injury (TBI) could lead to the development of molecular diagnostics which could be used to aid acute detection in scenarios where neuroimaging in unavailable, or to remotely track injury progression and recovery. It is becoming increasingly evident that brain tissue exhibits patterns of micro‐RNA (miRNA) expression which are distinct from those of other tissues. During TBI, the blood brain barrier becomes disrupted and molecules from damaged neural tissues are released into peripheral circulation. Thus, the detection of brain specific miRNAs in peripheral blood could serve as a surrogate marker of TBI. In this investigation, we aimed to systematically identify brain‐enriched miRNAs, and then test their potential utility for use as TBI biomarkers. First, we obtained publically‐available expression data for over 1,300 miRNAs generated from 31 different human post‐mortem tissues. Tissue specificity index (Tau) was calculated for each miRNA, and they were subsequently ranked in terms of their degree of brain‐specific expression. The abundances of the top five ranked miRNAs were then measured in serum samples collected from 10 TBI patients and 10 healthy controls using qPCR, and evaluated for their ability to discriminate between groups using k‐nearest neighbors (k‐NN). The top five miRNAs identified in our tissue specificity analysis consisted of miR‐137, miR‐219a‐5p, miR‐128‐3p, miR‐124‐3p, and miR‐138‐5p, which exhibited from 31 to 74‐fold higher expression levels in brain relative to other tissues. Three out of the five candidate miRNAs exhibited significantly higher abundance in serum samples from TBI patients relative to control patients in qPCR analysis, and their coordinate expression levels were able to discriminate between groups with 90% sensitivity and 80% specificity. The five candidate miRNAs identified in our analysis have potential utility for use as TBI biomarkers, and such a possibility warrants further investigation.

Support or Funding Information

Work was supported by Case Western Reserve University FPB School of Nursing start‐up funds issued to Grant C. O'Connell.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Toxicity and Immunogenicity of a Tardigrade Cytosolic Abundant Heat Soluble Protein
Authors: Esterly Harrison James , Crilly Candice , Piszkiewicz Samantha , Pielak Gary , Christian Brooke
Published: June 2021

Tardigrades are microscopic animals recognized for their resilience to extreme stresses, including desiccation, freezing, boiling, low and high pressures, and high doses of radiation. Their ability to survive desiccation is attributed to tardigrade‐specific cytosolic abundant heat soluble (CAHS) proteins. Expression of these proteins in other cells increases the desiccation tolerance of those species, and in vitro, CAHS proteins inhibit desiccation‐induced inactivation of other proteins. We are seeking to test the ability of CAHS proteins to stabilize protein‐based therapeutics, which require refrigeration for transportation and storage. The ability of CAHS proteins to preserve the activity of these pharmaceuticals upon desiccation at room temperature would increase their availability and reduce their cost. To this end, we investigated the toxicity and immunogenicity of CAHS D when injected into mice. Recombinant CAHS D was expressed in E. coli and purified by cation‐exchange chromatography. Purity was verified by mass spectrometry prior to endotoxin removal. Purified CAHS D was injected at various concentrations, and mice were monitored for symptoms of toxicity. No changes in weight or behavior were observed during the 28‐day monitoring period. A large‐scale toxicity study, as well as additional studies to test immunogenicity, is underway.

Support or Funding Information

University of North Carolina System Institutional Planning Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Squishing Cellular H2O
Authors: Bartolomei Barrett , Bogdon Phoebe , Frazer Mandy , Johnson Robert , Lopez Dayana , Mergens Steven , Nichols Andrew , Owens Jonathan , Rall Tanner , Rhodes Abigail , Shipley Ashley Willow , Simmons Emily , Wright Zachary , Galea‐Martinez Sandra Ann , Bresniker Catherine
Published: June 2021

Water is an essential part of the function of all living organisms. If water does not function normally, neurodegenerative and autoimmune diseases can become everyday occurrences affecting the health of humans. A research study was conducted about the structure and relative significance of the water channel, aquaporin 4 (AQP4), and it's possible conformational changes. Though structural details concerning the molecular mechanism of human AQP4 regulation are still unknown, this study has assisted in gaining insight into its structure and its interactions. The protein AQP4 consists of six‐transmembrane domains and five connecting loops that form a channel, and similar to other aquaporin channels, the monomers of AQP4 assemble into tetramers. AQP4 is namely arranged in clusters, known as orthogonal array particles, which allow for better anchoring to the membrane. Similar to other aquaporins, the pathway through the channel is amphipathic; this disposition allows for the bidirectional conductance of water. The physiological aspect (which is a 4‐fold axis structure) insulates against all solutes and water; the cytoplasmic side of the channel stabilizes the backbone amides. The mid‐membrane section, containing Phe‐195, Leu‐191, and Leu‐75, create a hydrophobic block (when repeated 4‐times). The research also revealed that AQP4 is subject to posttranslational regulation by phosphorylation. Water conductance is affected in AQP4 when SER‐111 (associated with the B loop) and Ser‐180 (associated with the D loop) are in a state of phosphorylation. Phosphorylated Ser‐180 is believed to possibly block the water channel by creating a tether to the C‐terminal domain. When Ser‐111 goes through phosphorylation, it mimics the gating mechanism in plants. This study of AQP4 suggests that the brain, spinal cord, and circadian rhythm are affected by its expression, as AQP4 is concentrated in the ends of astrocytes, of the brain. As new research comes to fruition, it is clear that these conformational changes will have broad implications with a variety of cell communication through that of water transport. The science community is on the frontier of research which could lead to therapeutic strategies and treatment of not only neurodegenerative diseases but autoimmune diseases as well. The El Capitan High School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of Aquaporin 4.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ras and RTK: PI3K Activation, PIP3 Formation, Signal Transduction, Cancer Creation
Authors: Chou Chris , Lubkeman Kelly , Martyr Richard , Adler Rose , All Caleb , Arroyo Jorge , Austin Colby , Daniel Graham , Fehlberg Ezra , Grassmick Emma , Greer Maya , Hak Iain , Hubbell Grayson , Olmstead Grant , Perez Annette , Roberts Lauren , Skaggs Erin , Tafoya Frankie Rose , Whitted Jackson , Falke Joseph J
Published: June 2021

Ras mutations are implicated in over 30% of all cancers. Under normal conditions, Ras becomes active by binding to guanine triphosphate (GTP) to stimulate cell growth and division and other cell pathways. When Ras acquires specific mutations, it no longer binds the GTPase‐activating protein (GAP), which impairs the GAP GTPase activity, trapping Ras in its active state and leading to uncontrolled cell proliferation. The Ras family of G‐proteins play a role in recruitment of phosphatidylinositol‐3‐kinase (PI3K) to the membrane and stimulation of PIP3 lipid signaling. The Ras family consists of 3 major isoforms: H‐Ras, K‐Ras, and N‐Ras; all having similar functions in different pathways. The primary sequence of amino acids among the Ras isoforms is highly conserved with differences existing primarily between their C‐terminal tails. The Falke lab is studying the H‐Ras variant. H‐Ras, in its active GTP‐bound state, and receptor tyrosine kinase (RTK) synergistically recruit PI3K to the membrane and stimulate PI3K kinase activity. During recruitment to the lipid bilayer, PI3K binds its substrate lipid phosphatidylinositol‐4‐5‐bisphosphate (PIP2) and also binds adenosine triphosphate (ATP) and then phosphorylates PIP2, creating phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). PIP3 then plays a central role in many cell growth and regulatory pathways by recruiting to the membrane many proteins that bind to its headgroup. Specific surface amino acid sidechains on Ras that bind to specific surface amino acid sidechains on the Ras‐binding domain (RBD) of PI3K during their interaction to assemble the fully active PI3K complex were modeled. Drugs that inhibit Ras or PI3K or their interaction may be effective in attacking cancer cell growth. Ultimately, understanding Ras and the mechanism of PI3K activation promotes understanding of a large subset of human cancers and facilitates the development of targeted treatments. The Longmont High School SMART (Students Modeling A Research Topic) Team modeled wild‐type H‐Ras and oncogenic mutants using 3D printing technology to investigate structure‐function relationships.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrated of Sigma Quanta Energy in Psycho‐Neuro‐Physio Dynamics Therapy for Symptoms & Cause of Osteoarthritis: A Case Study
Authors: Sainvil Frantz Lee , Einstein George P , Konyk Carla M , Tulp Orien L , Rahman Michael S
Published: June 2021

Osteoarthritis is the most common joint disease, afflicting some 27 million people in the United States alone. Over the past several decades, progress in the measurement of rates and interactions of molecular and cellular processes has initiated our understanding of dynamics phenomena of cells, Living cells are inherently dynamic. The vast qualitative information in the treatment of osteoarthritis is by allopathic medicine that can be upgraded by the individual algorithm of Sigma Quanta Energy in Psycho‐Neuro‐Physio Dynamics Therapy

Methods and Research

Generally, the phrase dynamics phenomena refers also to Psycho‐Neuro‐Physiotherapies. The research on human subjects prepared algorithm therapy and life style. Algorithm of psychotherapy‐ by daily 15 min meditation, 30 minutes of Acu‐yoga exercises and 15 minutes meditation after. The effect of psychological intervention was also on regression of

Algorithm of neuro therapy that emphasized nutrients for brain to support the threshold of the magnitude of stress intensity of an individual that must be exceeded for a certain reaction in meditation and Acu‐yoga exercise, phenomenon, result, condition to be manifested. Algorithm Physio‐Dynamics was design for application to microbial infection that gave a systematic delivery of complement inhibitors.

Results

The aim of this research was determining the effect of Integrated of Sigma Quanta Energy in Psycho‐Neuro‐Physio Dynamics Therapy for Symptoms & Cause of Osteoarthritis. The application of the Triple Algorithm Dynamic therapy has been demonstrated to diminish or eliminate the osteoarthritic pain. There is a need to take daily a holistic approach and consider the impact of combinations of stress reduction, meditation exercise and dietary patterns, with a likely greater overall benefit than each single component might have on its own.

Support or Funding Information

Supported by Institutional Resources of USAT, UHH, and EMI

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Upregulated miR‐144‐3p Attenuates LPS‐induced Acute Lung Injury via Inhibition of ROCK1 Signaling Pathway
Authors: Siddiqui Mohammad Rizwan , Tauseef Mohammad , Shahid Mohd. , Macdonough Kelli , Shanley Thomas
Published: June 2021

Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of Acute Lung Injury (ALI) and sepsis that may results in a pathophysiological condition characterized by alveolar flooding and pulmonary edema and subsequent hypoxemia. In lung ECs, activation of Rho associated kinase‐1 (ROCK1) phosphorylates myosin light chain (MLC), stress fiber formation and hyper‐permeability during ALI. The role of MicroRNA‐144 (miR‐144) has been well investigated in many human diseases including cardiac ischaemia/reperfusion‐induced injury, lung cancer and lung viral infection, however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated miR‐144 mediated mechanism in the protection of endothelial barrier function in bacterial wall lipopolysaccharide‐(LPS) induced lung injury model. By using transwell endothelial permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR‐144 effectively blocked human lung EC barrier disruption and hyper‐permeability in response to LPS. Furthermore, using a gain and loss of function strategy, overexpression of miR‐144 significantly decreased ROCK1 expression. Concomitantly miR‐144 inhibits ROCK1 mediated phosphorylation of MLC phosphatase and thus phosphorylation of MLC to counteract actin stress fibers formation in LPS activated human lung EC. Finally, in mice challenged with LPS, in vivo delivery of miR‐144 mimic via liposomes attenuated endotoxemia‐induced increased in lung wet‐to‐dry ratio, vascular permeability and inflammatory cytokines. In conclusion, our data suggest miR‐144 attenuates activation of the inflammatory ROCK1/MLC pathway in vascular EC is a promising therapeutic strategy to counter inflammatory lung injury.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stability Studies of Rooperol and Analogues by In Vitro Metabolism with HPLC/MS Detection
Authors: Bohanon Amanda L , Kerwin Sean M
Published: June 2021

African Potato, Hypoxis hemerocallidea, has a long history of use by indigenous people of South Africa to treat cancer and a variety of other diseases. Extracts from the corm contain the norligan bisglycoside hypoxoside, which is hydrolyzed in the body to yield the anticancer agent rooperol (1,5‐bis(3′,4′‐dihydroxyphenyl)pent‐1‐en‐4‐yne). Studies have shown that rooperol selectively inhibits the growth of several cancer cell lines. Phase I clinical trials of rooperol in lung cancer patients suggested promising activity with no dose‐limiting toxicity. However, rooperol is rapidly converted to biologically inactive sulfate and glucuronide metabolites. Thus, a relatively small amount of the drug reaches the tumor site. Bioisosteric analogues of rooperol have been synthesized with the aim of increasing metabolic stability while preserving the anticancer properties of the parent drug. The goal of this research is to investigate the metabolic stability of rooperol and analogues. As part of this study, we employ an in vitro metabolism assay with porcine liver microsomes. Microsomes are supplemented with the cofactor UDP‐glucuronic acid and the pore‐forming peptide alamethicin. The suitability of this assay to characterize the Phase II metabolism of phenolic compounds was established with the plant phytochemical 3‐hydroxytyrosol. The time‐dependent metabolism of this compound was determined by HPLC assay, and the formation of the glucuronide Phase II metabolite was confirmed by HPLC/MS. The time course for the disappearance of 3‐hydroxytyrosol followed first‐order kinetics with an apparent half‐life of 65.4 minutes. We have recently employed this assay to quantify the metabolic lability of rooperol. The disappearance of rooperol monitored by HPLC revealed an exceptionally short half‐life of 3.3 ± 0.59 minutes and an intrinsic clearance of 0.101 ± 0.02 mL/min/mg protein. The identities of glucuronide metabolites of rooperol were verified by HPLC/MS. The extreme metabolic lability of rooperol demonstrates the need to identify more metabolically stable analogues. We will discuss the integration of this in vitro metabolism assay in a work‐flow designed to identify rooperol analogues exhibiting increased metabolic stability while retaining the cytotoxic activity of rooperol.

Support or Funding Information

Robert Owens Medical Research Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chondroitin Sulfate Pharmacological Activity from Four Different Sources in Human Osteoarthritic Cartilage
Authors: Montell Eulàlia , Pelletier Jean‐Pierre , Martel‐Pelletier Johanne
Published: June 2021

Objective

Chondroitin sulfate (CS) is a glycosaminoglycan and a well‐known component of cartilage extracellular matrix, which has been shown to have clinical benefit in symptomatic osteoarthritis (OA) and data suggest to be a modulating agent of joint inflammation. CS is generally produced by means of extraction from different animal tissues (bovine, porcine and marine cartilage). Because of differences in CS in terms of source, purity, quality and the production/purification process, we compared the effects of CS from four different sources on the function of human OA chondrocytes.

Methods

Experiments were carried out on human OA chondrocytes obtained from knee patients undergoing total joint arthroplasty (n=8). Four different CS products were tested: CS#1, which is a high purity CS (98–100%) of bovine origin (Sioux Pharm Inc., Sioux Center, IA, US) and CS#2, CS#3 and CS#4, which are of lower purity and from other animal sources, CS#2 and CS#4, porcine; CS#3, avian. Determination of protein production of metalloproteinase (MMP)‐1 and MMP‐3, interleukin (IL)‐6 and prostaglandin E2 (PGE2) was performed on the conditioned medium using specific ELISAs. All determinations were performed in duplicate.

Results

CS#2 and CS#3 upregulated MMP‐1 (p=0.002, p=0.004) and CS#2 and CS#4, MMP‐3 (p=0.008, p=0.037). IL‐6 was also increased by CS#2, CS#3 and CS#4 (p=0.008, p=0.007, p=0.014). For PGE2 production, as the values were too low and unreliable without an inducer, the experiment was done only in the presence of IL‐1b (100 pg/ml), which greatly increased the value (absence of IL‐1b, 0.98±0.08 pg/mg protein; presence of IL‐1b, 42.18±10.80 pg/mg protein; p=0.007). CS#1 was the only one to significantly reduce PGE2 levels (p=0.035). In the presence of IL‐1b, CS#1 was also the only compound to show a trend toward significance (p=0.062) in reducing IL‐6 production.

Conclusions

Data showed that the three CS from non‐bovine origins increased an inflammatory factor in human OA chondrocytes, as well as upregulated the production of two catabolic factors. In contrast, CS of higher purity and coming from bovine source showed anti‐inflammatory properties as it reduced PGE2 and IL‐6 levels, even in the presence of IL‐1b. Therefore, the source/nature of these products could contribute to the overall biological and pharmacological effects of these agents in the symptomatic treatment of OA, compromising the safety and efficacy of the final products.

Support or Funding Information

Supported by BIOIBÉRICA, S.A.U., Barcelona, Spain.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vitro determination of the CB1 efficacy of illicit synthetic cannabinoids
Authors: Sachdev Shivani , Vemuri Kiran , Banister Samuel D. , Longworth Mitchell , Kassiou Michael , Santiago Marina , Makriyannis Alexandros , Connor Mark
Published: June 2021

BACKGROUND AND PURPOSE

The recreational use of synthetic cannabinoid receptor agonists (SCRA) new psychoactive substances is associated with significant morbidity and mortality compared with herbal cannabis. To explore the relationship between SCRA CB1 receptor activity and acute toxicity, we determined the CB1 efficacy of thirteen SCRAs using receptor depletion with the irreversible CB1 antagonist AM6544 followed by curve‐fitting with the Black and Leff operational model. We also investigated the effects of brodifacoum, an anticoagulant associated with a recent outbreak of SCRA poisoning, on the acute signalling of human CB1 receptors.

EXPERIMENTAL APPROACH

Receptor depletion in mouse AtT‐20 pituitary adenoma cells stably expressing human CB1 was achieved by pre‐treatment of cells with AM6544 (10 μM, 60 mins). The CB1‐mediated hyperpolarisation of AtT‐20 cells was measured using fluorescence‐based membrane potential dye. The efficacy (tau) and affinity (KA) parameters were obtained for each drug by fitting data to the operational model. We next determined the effect of brodifacoum (1 μM, 5 mins) on the hyperpolarization induced by subsequent application of CP55940 or Δ9‐THC.

KEY RESULTS

The tau value of Δ9‐THC was 80‐fold less than the reference CB‐agonist CP55940, and 260‐fold less than the highest efficacy SCRA, 5F‐MDMB‐PICA. The operational efficacy of SCRAs ranged from 233 (5F‐MDMB‐PICA) to 28 (AB‐PINACA), with CP55940 in the middle of the efficacy rank order. Application of brodifacoum had no effect on the potency, or the maximal response produced by high efficacy cannabinoid CP55940 or low efficacy phytocannabinoid Δ9‐THC (P<0.05).

CONCLUSION AND IMPLICATIONS

We determined the efficacy of a library of the most prevalent SCRAs identified in the new psychoactive substances market since 2008 and found that the SCRAs we tested had up to 300 times the efficacy of Δ9‐THC, which may contribute to the apparently greater toxicity of these drugs. Our data suggests that cannabinoid induced signaling was not affected by brodifacoum, indicating that combining SCRA with brodifacoum is not likely to enhance user experience through interactions with CB1.

Support or Funding Information

This work was supported by NHMRC Project Grant 1107088 awarded to M.K., and M.C. and National Institutes of Health grant P01DA009158 to A.M.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ZnR/GPR39 mediated human salivary secretion
Authors: Kim Yoon‐Jung , Jo Youhwa , Jo Su‐Hyun , Park Hee‐Kyung , Choi Se‐Young
Published: June 2021

Zn2+ is divalent cation that is essential for many biological events by affecting various ion channels. Zn2+ can serve G‐protein coupled receptor signaling via activation of metabotropic zinc receptors, ZnR/GPR39. In spite of several evidences of the existence of ZnR/GPR39 in salivary gland cells, there has been no evidence of ZnR/GPR39‐mediated modulation of the salivary gland functions. Here we have characterized the role of ZnR/GPR39 in human submandibular gland cells. Mouth wash with Zn‐containing solution evoked the secretion of unstimulated and stimulated whole saliva in human. We found that ZnR/GPR39 is expressed in the human submandibular glands and HSG cells. Zn2+ increased cytosolic Ca2+ concentration ([Ca2+]i) in a concentration‐dependent manner. Both muscarinic antagonist and histaminergic antagonists did not have any effect on Zn2+‐induced increase [Ca2+]i, which is completely blocked by the inhibitor of phospholipase C‐beta. Like as muscarinic and histaminergic stimulation, Zn2+ induced the translocation of aquaporin‐5 (AQP5) into the plasma membrane. These data suggest that metabotropic zinc receptors can modulate salivary secretion in human submandibular gland cells independently from the muscarinic receptor signaling.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (2017R1A2B4002176 to H.K.P.; 2018R1A5A2024418 to S.Y.C.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFFECT OF INTERFERON‐γ ON THE NEUTROPHIL FUNCTIONAL PROPERTIES
Authors: Duarte Ana Paula Ferreira , de Souza Ivani Aparecida , Ramos Verônica Santos , Torres Anelize Sgorlon Pinheiro , Antunes Edson
Published: June 2021

Interferon γ (IFN‐g) is a cytokine produced by natural killer T cells implicated in innate and acquired immune response during bacterial infections. Higher levels of this cytokine are suggested to suppress the hematopoiesis, leading to leukopenia that is clinically observed after the resolution of severe infectious. Evidence shows that IFN‐g induces MHC class II overexpression in human circulating neutrophils (NE) and reduces Staphylococcus aureus‐mouse induced NE infiltration. Our preliminary results have been showing that staphylococcal enterotoxins (SEs; type A and B) are able to inhibit human NE properties such as adhesion and chemotactic response. Whereas, IFN‐g release is reported to contribute to pathological events induced by SEs the aim of the present study was investigate the effect of IFN‐g on the functional properties of human NE. Blood was collected from healthy volunteers after approval from the local ethics committee (Protocol No 61370616.3.0000.5412). Blood samples were placed on isotonic Percoll solution. After centrifugation, the mononuclear cell layer was removed and the pellet containing erythrocytes and granulocytes was aspirated and subjected to isotonic lysis. After lysis cells (98% NE) were resuspended to 4 × 106 cells/ml and submitted to in vitro incubation with IFN‐g (10000 IU/ml; 30 min). Adhesion assays were carried out in 96‐well plates pre‐coated with recombinant human VCAM‐1 and ICAM‐1 for 30 min in the presence of interleukin‐8 (IL‐8). The NE adhesion was calculated by measuring myeloperoxidase (MPO) activity on adherent cells. NE migration assay was performed using a 48‐well microchemotaxis Boyden chamber. Prior NE in vitro incubation with IFN‐g reduces human NE adhesion in VCAM‐1 (Control: 4.7 ± 0.5; untreated NE + IL‐8: 6.7 ± 0.6; IFN‐g treated NE+IL‐8: 4.8 ± 0.66 optic density/NE × 106 cells; ) or ICAM‐1(Control: 4.3 ± 0.5; untreated NE + IL‐8: 6.1 ± 0.6; IFN‐g treated NE+IL‐8: 3.9 ± 0.48 optic density/NE × 106 cells) coated plates. Similar results were observed for NE chemotactic activity (Control: 18.7 ± 0.8; untreated NE +IL‐8: 52.9 ± 2.7; IFN‐g treated NE+IL‐8: 36.4 ± 1.6 NE per high‐power field). These findings support the hypothesis that IFN‐g modulates the inhibitory effects of Staphylococcal enterotoxins on NE functional properties.

Support or Funding Information

CAPES‐Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (1676187).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelin‐1 Induced Fibronectin Expression via c‐Jun
Authors: Hernandez Sergio J.r
Published: June 2021

Glaucoma is an optic disease that is characterized by slow neurodegeneration of the optical nerves and steady loss of retinal ganglion cells. Over 70 million people suffer from glaucoma and it is estimated that over 118 million will suffer from it in 2040. A prime factor causing glaucoma is an increased in intraocular pressure (IOP) but the mechanism in how IOP contributes to glaucoma is still being studied. Blocking the aqueous humor through the trabecular meshwork (TM) is a major contributor for the IOP elevation. Endothelin (ET‐1) is a potent vasoconstrictor in which play's an important role in vascular system. Previous research has discovered that ET‐1 and its ETA/ETB receptors are also involved in the pathogenesis of glaucoma. The current study aims to test whether ET‐1 induced fibronectin deposition at TM. We hypothesize that ET‐1 induces over expression of fibronectin through c‐Jun‐mediated signaling pathway. Primary human TM cells were pretreated with c‐Jun siRNA, SP600125 (a JNK inhibitor to block activation of c‐Jun) or ET receptor antagonist's followed with ET‐1 treatment. Protein levels of fibronectin and c‐Jun were detected using immunoblotting. An increase of fibronectin and c‐Jun were detected in TM cells treated with ET‐1 for 24–72 hours. Fibronectin was increased as a result of c‐Jun being partially terminated by the knockdown of c‐Jun by C‐Jun siRNA. Blocking of ETA/ETB by BQ610/BQ788 reduced the expression of fibronectin. Our results preliminary demonstrate that ET‐1 induces the expression of fibronectin through c‐Jun mediated signaling pathway, suggesting that ET‐1 may cause the accumulation of fibronectin at TM contributing to IOP elevation.

Support or Funding Information

This work was supported by the Department of Health and Human Services, National Institute of Health, National Heart, Lung and Blood Institute, SMART Award Number 4R25HL007786‐25 to Harlan Jones, Ph.D This work was also supported by MARC U*STARGrant5T34GM008395 to MariaElena Zavala, Ph.D

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐precision and scalable fabrication of biomimetic culture environments for enhancing adherent mammalian cell and tissue engineering
Authors: Ghazizadeh Hamed , Fisher Elliot , Gray Kevin M , Geisse Nicholas A
Published: June 2021

Cells maintained in vitro typically lose various structural and functional phenotypes that are found in vivo. Generation of biomimetic substrates typically involves costly or hard‐to‐reproduce techniques that are often incompatible with many standard assays. Here, we will present a novel method of generating surfaces that mimic the nanoscale shape and structure of the collagen ECM. The fabrication scheme described is highly reproducible, scalable, and amenable to integration with most end‐point assays, including high‐NA optical microscopy. Further, we present techniques to can fabricate biomimetic culture surfaces out of elastomers that can be stretched in order to reproduce mechanical stimuli that are critical in the development and function of some tissues. Various cell types were tested and were amenable to this approach. Cardiac tissues showed more in vivo‐like myofibril alignment, sarcomere spacing and width, and expression of CM‐specific proteins. Stem‐cell derived CMs similarly showed earlier onset of maturity and differentiation. We will show that the nanoscale patterning also maintains the migratory phenotype of metastatic cancer cells in vitro. Finally, we will demonstrate that this patterning approach can be extended to high‐throughput electrophysiological assays when combined with micro‐electrode arrays (MEAs) without compromising signal fidelity but enhancing sensitivities to drugs of known in vivo effect.

Support or Funding Information

NIH SBIR Funding

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.