bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemoproteomic Profiling of Cellular Substrates of the Lysine Acetyltransferase HAT1 Using Cell Permeable Bioorthogonal Reporters
Authors: Song Jiabao
Published: May 2022

Although quantitative proteomics studies have identified thousands of acetylation substrates and acetylation sites in both eukaryotic and prokaryotic cells, our knowledge of the acetylation scopes contributed by individual lysine acetyltransferases (KATs) is still very limited. A representative example is that functional studies on the histone acetyltransferase 1 (HAT1), one of the first KAT members identified in mid‐1990s, have been largely restricted on its nuclear histone substrate. Lacking information on the proteome‐wide substrate profiles of HAT1 and other different KAT members greatly hinders our understanding of functional roles of this important family of enzymes in regulating cellular physiology and pathology. Recently, acyl‐CoA reporters have emerged as effective tools to label KAT substrates in complex proteome settings, especially in whole cell lysates. However, it was commonly regarded that acyl‐CoA molecules are not cell‐permeable and cannot be applied for performing protein acylation profiling in living cells. Here, we report that a clickable acyl‐CoA analog, 3‐azidopropanoyl CoA (3AZ‐CoA), showed remarkable cell permeability and effectively labeled proteins in cellulo. We rationally engineered the active site of HAT1 by site‐directed mutagenesis and obtained a HAT1 mutant that showed excellent bioorthogonal activity utilizing 3AZ‐CoA as a cofactor to effectively acylate and probe HAT1 substrates. Importantly, we were able to apply the bioorthogonal enzyme‐cofactor pair to achieve HAT1 substrates labeling, enrichment, and identification from the living cells. The reactive azide warhead group successfully allowed for the labeled protein substrates to be pulled down following the chemoselective conjugation with the biotin handle through the copper‐catalyzed alkyne‐azide cycloaddition chemistry. Subsequent SILAC proteomic analysis of the bioorthogonally enriched proteins revealed hundreds of novel HAT1 substrates, which include proteins critical for transcription, RNA splicing, mitochondria, metabolism, and oxidation‐reduction process pathways. These findings underline that HAT1 has broader functionalities in mammalian cells than previously thought. The methodology we applied herein also proves that cell‐permeable acyl‐CoA reporters that orthogonally match with corresponding KAT enzymes forms serve as a powerful chemical biology tool for functional mapping of the acetylomes of different KAT isoforms from their native biological contexts.
Postnatal Phrenic Motor Neuron and Diaphragm Maturation in the Mouse
Authors: Brandenburg Joline E. , Fogarty Matthew J. , Davis Leah A. , Sieck Gary C.
Published: May 2022

In rats, there is a strong association between the expansion of phrenic motor neuron (PhMN) size (from ~21‐28 days postnatal), the emergence of mature diaphragm muscle (DIAm) myosin heavy chain (MyHC2X and MyHC2B) isoform expression, alongside the manifestation of mature morphological and functional properties. Despite mice models being de rigueur for developmental investigations, the postnatal development of mouse PhMN and DIAm properties have not been comprehensively characterized. Using wildtype C57 mice at ages P7, P14, P21, P28, and >3 months (maturity) we evaluated PhMN and DIAm physiologic properties. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 µm) and prepared for confocal imaging and PhMN assessment. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh‐frozen, with 8 µm cross‐sections immuno‐labelled for MyHCslow, MyHC2A, MyHC2X and MyHC2X and processed for fluorescent assessment. The number of PhMNs remained stable throughout postnatal life (~195), while PhMN surface area increased steadily, ~10% from P14 to P21, P21 to P28, and P28 to maturity, with overall ~30% increase from P14 to maturity. The biggest changes in number of primary dendrites occurred between P14 and P28 and between P28 and maturity, 25% decrease in primary dendrites between each group. DIAm maximum Po increased in two phases, 3.5‐fold from P7 to P14 and remained similar until P21. The second large increase was ~3‐fold from P21 to P28 and remained steady to maturity. The fatiguability of the DIAm increased with age in two phases, with fatigability being similar from P7 and P14 and increasing by ~20% at P21. Following, fatigability of the DIAm was similar from P21 to P28 and increased by ~35% at maturity. These major PhMN and DIAm changes coincide with the steady growth of PhMN and the emergence of MyHC2X and MyHC2B expression in DIAm from P14 to P21 onwards. The maturation of orderly PhMN recruitment likely depends on an interplay between PhMN size properties and network inputs to a refined dendritic tree. We show that DIAm muscle fiber maturation in mice hinges on the expression of MyHC2X and MyHC2B isoforms that is associated with an ~10 fold increase in DIAm force‐generation capacity.
The Pandemic Effects on the Microbiome of Infants in the Neonatal Intensive Care Unit (NICU)
Authors: Xia Jason S. , Oliphant Kaitlyn , Claud Erika
Published: May 2022

The microbiome is an *invisible organ* that affects health and disease. Early life microbial colonization is critical for development of the immune system, postnatal growth, and long‐term health. The dynamic and nascent microbiomes of children are highly individualized. SARS‐CoV‐2(Covid‐19) is known to impact the adult microbiome. However, pandemic impact on the infant microbiome is unknown. In the current study we hypothesized that infants born prior to pandemic regulations may demonstrate differences compared to those born during the pandemic. Fecal samples (n=307) were collected from infants (1‐19 weeks) in the NICU at The University of Chicago. DNA was extracted for 16S rRNA sequencing, and 4576 taxa were analyzed using R. We performed alpha diversity analysis within the samples and beta diversity between the samples to measure and compare bacterial communities. Alpha diversity did not demonstrate a significant difference between before pandemic and during pandemic groups, while Beta diversity did achieve a significant difference between the two groups. Multivariate analysis showed that in before pandemic and during pandemic groups, post menstrual age (PMA), weeks of life (WOL), as well as the interactions of pandemic grouping by PMA, and PMA by WOL were significant. Lastly, we identified the top 5 positively associated and top 5 negatively associated bacteria at the species/genus level associated with the pandemic grouping. Of these, Serratia marcescens and Acinetobacter johnsonii are associated with antibiotic resistance, and reduction of human pathogens such as Shigellaand Salmonella.This study illustrates that differences in microbiome diversity exist between infants before and after pandemic regulations. Any disruptions in microbial colonization can contribute to shifts in normal microbial colonization that persist past the first 1000 days of life. Our study suggests the possible influences of secondary factors during the pandemic on microbiome development. In future studies we plan to further explore the interaction of cofounders (e.g. PMA) as well as follow up on the long‐term effects of the pandemic on the infant microbiome.
Characterization of Protein Tyrosine Phosphatase 1B activator peptides in cells
Authors: Londhe Avinash D. , Bergeron Alexandre , Rizvi Syed H. , Kim Seung J. , Boivin Benoit
Published: May 2022

The Protein Tyrosine Phosphatase (PTP) family of phosphatases regulates cell signaling through dephosphorylation of serine, threonine and tyrosine residues of downstream effector proteins. The role of PTPs is very well known in normal cell functions, as well as in disease states such as diabetes and cardiovascular diseases. We previously reported on the role of the phosphotyrosine recognition loop (PTBL) of PTP1B in protein‐protein interactions (Londhe et al.2020, Nat. Chem. Biol., 16(2):122‐125) and showed that interfering with protein‐protein interactions between PTP1B and 14‐3‐3zusing R18, a pan‐14‐3‐3 inhibitor, destabilizes the reversibly oxidized form of PTP1B (PTP1B‐OX) and activates the phosphatase. We rationally designed peptides using phosphotyrosine recognition loop of PTP1B to determine whether selective and effective activation of PTPs was possible. We first performed a structural analysis to compare the changes occurring in PTP1B‐OX compared to its reduced form. Using this approach, we observed profound conformational changes in the phosphotyrosine recognition loop (PTBL) of PTP1B and rationally designed peptides comprised of the sequence of amino acids of the newly exposed loop, with a TAT (transactivator of transcription) peptide sequence in amino or carboxy‐terminal to facilitate cell permeability. Targeting the PTP1B‐OX‐14‐3‐3z interaction using R18 and TAT‐PTBL peptide led to disruption in protein‐protein interaction and maintained PTP1B in its active form. Interestingly, destabilizing the PTP1B‐OX form by treating cells with rationally designed PTP1B activator peptides also decreased EGFR phosphorylation at PTP1B sites. In cells stimulated with exogenousH2O2 to bypass the NOX‐mediated PTP1B oxidation, the pretreatment with R18 and TAT‐PTBL prevented the interaction between PTP1B‐OX and 14‐3‐3z. Then we observed that our TAT‐PTBL peptide was not interfering with NOX‐mediated reactive oxygen species (ROS) generation, whereas R18, a pan‐14‐3‐3 inhibitor, decreased ROS production significantly. Finally, exposing A431 epidermoid carcinoma cells to PTP1B activator peptides prevented EGFR phosphorylation and impaired colony formation. Collectively our data suggest that the biological activity of PTP1B activator peptides is likely to be therapeutically useful, and could translate into drug leads.
Student created quizzes to reinforce critical information and highlight deficits for both the students and instructor
Authors: Patenaude Bart , Scott Joanna
Published: May 2022

Introduction

Medical students use passive strategies to study even though evidence shows the benefits of active learning. Having students work in groups and write quiz questions is an effective way to engage the students in active learning.

Methods

Students in a dental head and neck anatomy course worked in groups of 7‐11 and created review quizzes for extra credit. The quizzes covered the material in the module and were posted a week before the module’s exam. In addition to the posted quiz, they posted the correct answers and a justification for why they were asking the question.

Results

104/109 (95%) students agreed to be part of the educational research. The majority of students thought that the writing of questions and course structure improved their educational experience (81%). The strength of this opinion increased from the first exam to the final (p<0.001).

Discussion

Using a small amount of extra credit points to motivate students to create quizzes was impactful on the students learning. It also was appreciated by them. It could be added to a course without any additional resources and without removing any material from the course. Finally, it gave the instructor a valuable window into the students’ understanding of the material taught.
Development of a gel filtration protocol that eliminates precipitation during refolding of chemically denatured proteins
Authors: Odunuga Odutayo O. , Tovar Carmel N.
Published: May 2022

Purification of recombinant proteins in denaturing conditions has many practical advantages. Chemical denaturants such as urea and guanidine hydrochloride are used to solubilize recombinant proteins from inclusion bodies. Proteins purified in denaturing conditions generally contain fewer contaminants compared to their counterparts purified in native buffers. Denatured proteins can be stored for long and refolded as needed. Refolding is commonly performed by equilibrium dialysis and sometimes on metal affinity or gel filtration columns by diluting out the denaturant and allowing the protein to fold back into its native state. The refolding steps are often tedious and result in significant loss of protein through aggregation. Using a modified gel filtration technique, we successfully refolded a spectrum of proteins that either are highly aggregation‐prone or contain several disulfide bonds or both, back to their native states. Some of the proteins are well known to precipitate significantly during refolding. However, using this technique, precipitation was either minimal or non‐existent. Structural analyses using circular dichroism and fluorescence spectroscopies, gel filtration, and coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins, and simultaneously performing buffer exchange and additional purification steps.
Treatment of Sleep Disordered Breathing with Leptin Loaded Extracellular Vesicles
Authors: Freire Carla , Pho Huy , Ramsey Jacob D. , Zhao Yuling , Kim Lenise J. , Berger Slava , Anokye‐Danso Frederick , Sennes Luiz U. , Ahima Rexford S. , Batrakova Elena V. , Kabanov Alexander V. , Polotsky Vsevolod Y.
Published: May 2022

Introduction

Obstructive sleep apnea (OSA) is the most common type of sleep disordered breathing (SDB), characterized by recurrent periods of upper airway obstruction during sleep leading to increase in morbidity and mortality. The prevalence of OSA exceeds 50% in individuals with obesity and 10‐20% also develop hypercapnia and hypoventilation during sleep which characterizes obesity hypoventilation syndrome (OHS). There is no effective pharmacotherapy for SDB. Leptin, an adipocyte derived hormone, can stimulate breathing and is a potential therapeutic candidate. However, diet‐induced obesity is associated with impairment of leptin transport across the blood‐brain barrier. Previous studies have shown that extracellular vesicles (EVs) naturally derived from macrophages can penetrate the BBB and be used for drug delivery. The objective of this study was to determine if EVs overcome the BBB and treat SDB in DIO mice.

Methods

To examine the ability of EVs to cross the BBB, lean (n=5) and DIO (n=5) C57BL/6J mice were injected with fluorescent EVs or saline into the lateral tail vein. After 4h EV biodistribution was evaluated by Bioimaging and Infrared Spectroscopy (IVIS). Sleep studies were performed in a plethysmography chamber in a separate subgroup of male, DIO (n=10) and lean (n=10) mice. Mice received saline, empty exosomes, free leptin, or leptin‐loaded exosomes in a crossover manner. Similar design was used to determine energy expenditure in a Comprehensive Laboratory Animal Monitoring System (CLAMS).

Results

We demonstrated that EVs accumulated in the brain and the transport across the BBB was 2‐times more efficient in DIO when compared to lean mice (p<0.005). In DIO mice, leptin EVs induced 1.7‐2.2‐fold increases in minute ventilation and 1.5‐2.0‐fold increases in maximal inspiratory flow during flow‐limited and non‐flow limited breaths (p<0.05). In contrast, free leptin had no effect. There was no effect of leptin EVs on metabolism. These findings suggest that leptin EVs mitigated upper airway obstruction and improved control of breathing in DIO mice. Lean mice did not present significant sleep disordered breathing and no differences were observed between groups.

Conclusion

We demonstrated that EVs overcome the BBB and that leptin loaded EVs treat SDB in DIO mice.
A dual potassium channelopathy underlies small vessel disease of the brain in a mouse model of Alzheimer's Disease
Authors: Taylor Jade , Pritchard Harry , Walsh Katy , Hennig Grant , Allan Stuart , Nelson Mark , Greenstein Adam
Published: May 2022

Mounting clinical and basic evidence has suggested that cerebral microcirculatory dysfunction is key to the pathogenesis of Alzheimer’s disease (AD), thus posing a potential new therapeutic target for AD treatment. However, despite observations of altered cerebral blood flow and autoregulatory capacity in AD patients, the underlying pathogenic mechanisms behind these defects are currently unknown. Here we present the first detailed study in to vascular ion channel function in the APP23 mouse model of AD. This model has a seven‐fold increase in amyloid precursor protein leading to substantial amyloid‐β plaque accumulation within the brain, as is described in AD patients. Using a range of physiological techniques; mainly pressure myography, patch clamp electrophysiology and high‐speed spinning disc confocal microscopy, we show that attenuation of large conductance calcium–activated potassium (BK) channel function in this model, due to a reduced frequency of calcium sparks, is responsible for the reduction in cerebral blood flow described. Further investigation into the role of amyloid‐β 1‐40, in particular, showed that vessels exposed to the peptide for 30 minutes exhibited significantly reduced BK channel function at both the cellular and vascular level. However, disparate from the APP23 mouse model, acute exposure to amyloid‐β 1‐40 resulted in an increase in calcium waves. Both a decrease in calcium sparks (as seen in the APP23 model) and an increase in calcium waves (as seen with acute application of the amyloid‐β 1‐40 peptide) results in contraction of cerebral arteries and a subsequent reduction of cerebral blood flow. The data directs future research in to preventative strategies that may help to stop alterations in calcium event initiation, restore microvasculature function and promote a healthy brain environment to limit disease progression in AD patients.
Structure and drug development of the human formylpeptide receptors FPR1 and FPR2
Authors: Wang Lei , Zhuang Youwen , Sun Dapeng , Xu Eric H. , Zhang Cheng
Published: May 2022

The formylpeptide receptors (FPRs) mediate pattern recognition of formylated peptides derived from invading pathogens or mitochondria from dead host cells. They can also sense other structurally distinct native peptides and even lipid mediators to either promote or resolve inflammation. Pharmacological targeting of FPRs represents a novel therapeutic approach in treating inflammatory diseases. However, the molecular mechanisms underlying FPR ligand recognition are elusive. We report cryo‐EM structures of Gi‐coupled FPR1 and FPR2 bound to a formylpeptide and Gi‐coupled FPR2 bound to two synthetic peptide and small‐molecule agonists. Together with mutagenesis data, our structures reveal the molecular mechanism of formylpeptide recognition by FPRs and structural variations of FPR1 and FPR2 leading to their different ligand preferences. Structural analysis also suggests that diverse FPR agonists sample a conserved activation chamber at the bottom of ligand‐binding pockets to activate FPRs. Based on our structures, we further screen several new chemicals as potential drugs. Our results provide a basis for structure‐based drug design on FPRs.
Structural Studies of Copper‐Binding Proteins Encoded by the ycnOperon from Bacillus subtilis
Authors: Fisher Oriana S. , Guo Jing , Singh Aarshi N. , Damle Madhura S. , Szalai Veronika , Peters Stephen C.
Published: May 2022

Most living organisms encode an ensemble of protein transporters and chaperones to regulate copper levels. The ycn operon of the bacterium Bacillus subtilisis regulated in response to copper, suggesting that the three proteins it encodes may engage in direct interactions with copper ions to facilitate transport and homeostasis of this transition metal. Here, we use a combination of structural, biochemical, biophysical, and bioinorganic approaches to determine the structures of individual proteins and their interactions with copper and other binding partners. The YcnI protein encoded by the operon contains a domain of unknown function (DUF1775) at its N‐terminus. We determined the structure of this domain and characterized its interactions with copper ions, finding that it coordinates the metal using a unique “mono‐histidine brace” site that is highly conserved across other members of the DUF1775 family. Our data also suggest a potential role for this domain to serve as a chaperone to facilitate transfer of metal ions. Furthermore, we also identify potential copper binding sites within the transcriptional regulator encoded within the same operon. Together, these studies reveal new insights into the structure and copper‐binding properties of proteins encoded by the ycnoperon, providing additional perspectives on how they may work together to regulate copper homeostasis. As homologs of these proteins are shared by many other bacterial species, it is likely that analogous mechanisms exist for copper uptake and homeostasis beyond B. subtilis.
Catfish Fraction B preparation improves the testicular morphology and functions through modulating the apoptotic pathway in STZ‐induced diabetes rat model
Authors: Renno Waleed M. , AlHassan Jassim M.
Published: May 2022

Diabetes Mellitus (DM)‐induced reproductive dysfunctions and infertility in men are globally on the rise with the increased incidence of DM patients. Diabetes causes testicular damage leading to reproductive dysfunctions. Further, DM‐induced reproductive dysfunctions are multifactorial in etiology encompassing both central (neurogenic) and peripheral (vasculogenic) components and appear more severe and more resistant to treatment than nondiabetic reproductive dysfunctions. Thus, there is a need for newer, more effective therapies based on an increased understanding of the underlying mechanisms of DM‐induced reproductive dysfunctions. We have recently shown that Fraction B (FB) preparation from a catfish (Arius bilineatus, Val.) significantly lowered blood glucose, increased insulin levels, and caused regeneration of damaged organs and tissues in STZ‐induced diabetes in rats. This study investigated the effect of daily IP administration of 3 mg FB/kg animal body weight for 8 and 12 weeks on the histology and function of the testes and analyzed its role on the apoptotic pathway using STZ‐induced diabetes in male rat model. Our results showed that FB treatment significantly lowered the blood glucose and restored the sperm count and motility in the treated diabetic rats. Concomitantly, the treated diabetic animals showed significant recovery in the diameter and thickness of the reproductive epithelium of the seminiferous tubules with significant improvement in stereological parameters (Johnsen’s Scoring) of the testicular histology to close to control values. Further, the diabetic‐treated animals showed significant downregulation of pro‐apoptotic parameters and upregulation of anti‐apoptotic markers. In conclusion, our results revealed a positive therapeutic effect of FB preparation on testes following the 8‐ and 12‐weeks treatment of STZ‐induced diabetes in rats. This study opens the way for the first time to the possible therapeutic application of FB preparation to DM–induced reproductive dysfunctions and explores its possible curative mechanisms in diabetic testes.
Contrasting Metabolic Profiles of TGFβ2 and TNFα in the Induction of Retinal Epithelial‐Mesenchymal Transition (EMT)
Authors: Shu Daisy Y. , Frank Scott I. , Karg Margarete M. , Butcher Erik R. , Nnuji‐John Emmanuella , Shah Rishi , Gollapalli Deviprasad , Saint‐Geniez Magali
Published: May 2022

Epithelial‐mesenchymal‐transition (EMT) is a process whereby epithelial cells transdifferentiate into matrix‐producing mesenchymal cells. EMT of retinal pigment epithelial cells (RPE) plays a crucial role in severe ocular pathologies including retinal fibrosis. Emerging evidence implicates metabolic and mitochondrial dysfunction as key drivers of EMT. Both transforming growth factor‐beta 2 (TGFβ2) and tumor necrosis factor‐alpha (TNFα) have the capacity to induce EMT in RPE; however, little is known about their impact on mitochondrial metabolism. We hypothesized that TGFβ2 and TNFα should share similar metabolic effects, i.e. repressed mitochondrial function and enhanced glycolysis, to support EMT of RPE. Matured human primary RPE (H‐RPE) were treated with TGFβ2 or TNFα (both at 10 ng/ml) for up to 5 days for Seahorse metabolic assays (Mito and Glycolytic Stress Tests), qPCR and untargeted metabolomics analysis (West Coast Metabolomics Center) analyzed using MetaboAnalyst. Both TGFβ2 and TNFα induced the upregulation of mesenchymal genes (fibronectin and matrix metalloproteinase‐2) in H‐RPE. While TNFα significantly upregulated inflammatory genes (IL‐6, IL‐8, IL‐1β, MCP‐1), no changes were evident in TGFβ2‐treated cells. Metabolically, TGFβ2 induced the “Warburg effect” in H‐RPE with enhanced glycolysis and a suppression of oxidative phosphorylation capacity. Surprisingly, TNFα showed the opposite effect with enhanced oxidative phosphorylation (higher basal levels of oxygen consumption and maximal respiration) and reduced glycolysis and glycolytic capacity. Metabolomics profiling showed distinct metabolomics signatures associated with TGFβ2‐ and TNFα‐treated primary RPE cells. Of the 188 metabolites tested, 7 metabolites were significantly changed in TGFβ2‐treated cells and 26 in TNFα‐treated cells. Enrichment analysis depicted altered glutathione metabolism, proline metabolism, aminoacyl‐tRNA synthesis, nicotinamide metabolism, pentose phosphate pathway and arachidonic acid metabolism in TNFα‐treated cells whereas TGFβ2‐treated cells showed perturbations in the biosynthesis of fatty acids, tryptophan metabolism, linoleic acid metabolism and arachidonic acid metabolism. The divergent bioenergetic rewiring governing TGFβ2 and TNFα in their induction of EMT may be linked to their differential effect on inflammation. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences will enable the development of more effective drug targets for combating retinal fibrosis.
CBD formulation improves energetic homeostasis in dermal fibroblasts from Gulf War Illness patients
Authors: Zuniga Hertz Juan Pablo , Chiteti Ramamurthi , Pavlovsky Arthur , Golomb Beatrice
Published: May 2022

Gulf War Illness (GWI) corresponds to an array of symptoms that includes chronic fatigue, musculoskeletal pain, cognitive dysfunction, sleep disturbance, gastrointestinal, respiratory, and dermatological symptoms that affect ~250,000 U.S. military veterans that served in Operation Desert Storm/Desert Shield (1990‐1991). Mitochondrial function impairments have been shown in GWI patients. GWI patients report partial amelioration of chronic fatigue and brain fog after medicinal marijuana and CBD oils. Interestingly, cannabidiol (CBD) modulates mitochondrial physiology though this has not been characterized in detail. We hypothesize that GWI mitochondrial pathology can be recapitulated in dermal fibroblasts (DF) from subjects to help define and develop a cell‐based model to study GWI and CBD treatment of DF promotes energy production by improving mitochondrial physiology. GWI patients (gender/age matched to healthy controls) were recruited to collect skin punch biopsy explants that were processed and cultured in DMEM FBS 10% for 30‐days to obtain dermal fibroblasts. DF were treated with serial dilutions of Verséa™ CBD (50mg/mL) lipid formulation (VESIsorb® that increases 4.4‐fold Cmax). Using real time mitochondrial analysis by Seahorse, energy phenotype and mitochondrial function was analyzed in control and GWI DF. Mitochondrial networks and ultrastructure were studied by live‐imaging using MitoTracker™ and transmission electron microscopy, respectively. Energy phenotype studies suggest that GWI DF present with lower mitochondrial metabolism and higher glycolytic activity, compared with controls. Additionally, mitochondrial stress suggests a significant reduction in mitochondrial maximal capacity. Such data establish GWI derived DF as a personalized model system to study mitochondrial pathology in GWI. After 18h treatment with Verséa™ CBD, GWI DF show a significant improvement in mitochondrial and glycolytic metabolism; control patients show no increases in mitochondrial metabolism but improved glycolysis. Verséa™ CBD treatment induced mitochondrial networks re‐organization in DF. These findings suggest that CBD improves GWI DF mitochondrial physiology, thus improving energy homeostasis. Our data provide new evidence that will validate the potential of cannabinoids as a therapeutic strategy to mitigate energy imbalance that may contribute to detrimental symptomatology (i.e., chronic fatigue, brain fog, cognitive dysfunction, etc.) in GWI patients.
Vascular Endothelial Growth Factor is Augmented in the Kidney with Chronic Partial Ureteral Obstruction and Further Stimulated by Lipopolysaccharide
Authors: Ma Hongbao , Yang Yan , Khan Muhammad B.
Published: May 2022

Vascular endothelial growth factor (VEGF), a critical mitogen for angiogensis, has been shown to be activated in the inflammatory process of fibrotic lung disease and inflammatory arthritis. In the present study we examined the expression of VEGF in Sprague‐Dawley rats subject to chronic partial ureteral obstruction (PUO) for 30 days to produce renal interstitial fibrosis. To create PUO, the right kidney was removed and the left ureter diverted into the psoas muscles. A sham operation was performed in the control group. In an additional group of PUO, the inflammatory response was augmented by intraperitoneal injection of lipopolysaccharide (LPS) (1 mg/kg BW) 24 hr before removal of the left kidney to examine renal histology and VEGF expression by immunoblot and RT‐PCR analyses. In PUO rats intense inflammation and tubulointerstitial fibrosis were seen on histology examination. Immunoblot analysis showed that VEGF/b‐actin in the renal cortex was 10.1±1.1 (mean±SD) (densitometry units) in 8 PUO rats, greater than 5.3±0.7 in the 8 sham‐operated rats (p<0.05), and increased further to 12.5±2.7 in 8 PUO rats receiving LPS (p<0.05). VEGF/b‐actin in the medulla of the PUO kidney was 9.6±1.8, greater than 5.5±1.2 in the sham‐operated rats (p<0.05) and increased to 13.1±1.6 in PUO rats receiving LPS (p<0.05). VEGF mRNA expression in the cortex increased by over two‐fold (2.17±0.28, p<0.03) in PUO rats compared with sham‐operated rats and increased further in PUO rats receiving LPS (fold change of 3.14±0.29, p<0.01 vs PUO). VEGF mRNA expression in the medulla increased by 3.15±0.43 in PUO rats compared with sham‐operated rats (p<0.03) and increased further by 5.09±0.47 in PUO rats receiving LPS (p<0.03 vs PUO). These results show that VEGF expression is augmented in chronic obstructed kidney and further stimulated by LPS, suggesting that it participates in chronic renal tubulointerstitial fibrosis.
Mother with Obesity Induced by High‐Fat Diet Impaired Autophagic Process and Induced Renal Lipid Accumulation in the Offspring
Authors: Phengpol Nichakorn , Promsan Sasivimon , Sutthasupha Prempree , Lungkaphin Anusorn
Published: May 2022

Introduction

The prevalence of obesity has increased worldwide, and more women are entering obese pregnancy. The offspring born to maternal obesity have an increased risk of obesity and insulin resistance in later life and could affect offspring’s kidney injury. The abnormal renal lipid metabolism leads to renal lipid accumulation and these suggested to be the importance mechanism to develop and progress of chronic kidney disease (CKD). Lipophagy is the autophagic process to hydrolyze lipid droplet into free fatty acid for preserve energy and prevent an excess of lipid accumulation. The defect in lipophagy regulation is associated with renal lipid accumulation and contribute to kidney injury. Therefore, we hypothesized that maternal obesity induced by high‐fat diet programmed to increase offspring renal lipid accumulation via dysregulation of autophagic process and abnormal renal lipid metabolism leading to offspring’s kidney injury.

Methods

Female C57BL/6 mice were divided into two groups, control lean mother and maternal obesity groups, fed with standard and high‐fat diet, respectively, for 8 weeks. After that, female mice were mated with normal male mice and were housed and maintain each diet throughout gestation and lactation periods. After weaning, male offspring from each mother were sacrificed and blood samples and kidney were collected to evaluate autophagic and lipid metabolism.

Results

Maternal obesity was significantly increased body weight, fat weight, blood glucose and HOMA‐IR in the offspring. The increasing in renal cholesterol and triglyceride together with upregulated FAS, Srebp1, ADFP and CD36 protein expression indicating increased renal lipid synthesis and renal lipid uptake were observed in the offspring born to maternal obesity. Meanwhile, maternal obesity affected to downregulated PPARα and CPT1A demonstrating decreased renal lipolysis leading to exacerbated renal lipid accumulation in the offspring born to maternal obesity. The significant increase in mTOR, decrease in AMPK, Beclin‐1, LC3B and LAMP2 protein expression were shown in offspring’s kidney born to maternal obesity. These represented the inhibition of autophagic process, impaired autophagosome formation along with aberrant lysosome formation.

Conclusion

Maternal obesity affected to program offspring obese‐insulin resistance and increased the risk of kidney injury. The stimulation of renal lipogenesis and the suppression of renal lipolysis led to increased renal lipid accumulation. Moreover, maternal obesity induced impaired autophagic process by decreasing autophagosome formation and impaired lysosome formation. These alterations reduced the hydrolyzation of lipid droplet in the offspring’s kidney leading to more severity of renal lipid accumulation and resulting in offspring’s kidney injury.
Crotonate sensitizes IAPi in the disruption of latent HIV by modulating the ncNF‐κB signaling pathway at the step of p100 cleavage into p52
Authors: Jiang Guochun , Li Dajiang , Falcinelli Shane D. , Wong Lilly M. , Tang Yuyang , Browne Edward P. , Archin Nancie M. , Margolis David M.
Published: May 2022

Background

Antiretroviral therapy (ART) effectively suppresses HIV‐1 (HIV) replication to an undetectable level in the periphery; however, the treatment is life‐long and HIV reservoirs cannot be eliminated by ART alone in people living with HIV (PLWH). The major obstacle that impedes HIV eradication in ART‐treated PLWH is the latent viral reservoir harbors in the resting CD4+ T cells. Recently, the activation of non‐canonical (nc) NF‐κB pathway via IAP inhibitor (IAPi) or the SMAC mimetic (SMACm) compounds has raised as an attractive mechanism to disrupt HIV from latency. IAPi/SMACm targets IAPs to activate NF‐κB inducing kinase (NIK) followed by phosphorylation of IkappaB kinase (IKK), leading to the cleavage of p100 into p52. The latter forms a complex with RelB to stimulate transcription of its target genes after translocation into the nucleus. Previously, we discovered that crotonylation stimulates HIV latency reversal that elicited by PKC/NF‐κB agonist. We hypothesize that there may exist a functional interaction between protein crotonylation and ncNF‐κB signaling, which may enhance IAPi‐induced latency reversal to optiomally disrupt HIV latency for a cure of HIV.

Methods

Multiple HIV latency models were used, including Jurkat cell derived HIV latency models, the U1 promonocytic model of latency, the primary CD4+ T cell model of HIV latency and the resting CD4+ T cells isolated from ART‐suppressed PLWH. HIV expression was determined by flow cytometry and/or real‐time PCR. Protein expression and cleavage was determined by Western blot.

Results

We found that IAPi/SMACm can disrupt latent HIV. However, its ability to reactivate latent HIV varied among different HIV latency models, with a lowest efficacy in the primary CD4+ T cell model of latency. Interestingly, IAPi/SMACm‐induced disruption of latent HIV was greatly enhanced in the presence of crotonate in all these in vitro and ex vivo models. This was possibly mediated by the enhancement of p100 cleavage into p52 in the ncNF‐κB signaling pathway.

Conclusions

We propose that a blockage of p100 cleavage prevents an efficient activation of ncNF‐κB signaling pathway in the latently infected CD4+ T cells in PLWH, which could be relieved by targeting IAP proteins to induce p100 cleavage for the activation of ncNF‐κB signaling. Crotonylation, which was recently defined by us as a new epigenetic mark for the active HIV transcription, potentiates the reactivation of HIV from latency by further releasing the blockage at the p100 cleavage step, thereby enhancing ncNF‐κB activation to disrupt HIV latency. These data indicate that crotonylation could be exploited to develop novel strategies for HIV cure or remission in the future.
T Cells Contribute to Lipid Deposition in Male and Female Dahl Rats Fed a High Saturated Fat Diet
Authors: Ramirez Lindsey A. , Snyder Elizabeth E. , Mattson David , Abais‐Battad Justine , Dasinger John Henry , Sullivan Jennifer C.
Published: May 2022

Background

The Dahl rat has been used as a model of high fat diet (HFD)‐induced hypertension and adiposity. Genetic deletion of T cells blunts HFD‐induced hypertension. However, it is not clear if deletion of T cells can also reduce the adiposity caused by the HFD. T cells have been shown to play a role in many inflammatory conditions which are characterized by dyslipidemia. Therefore, the goal of the current study was to test the hypothesis that deletion of T cells prevents lipid deposits in the liver and adipose tissue.

Methods

5‐week‐old male and female wildtype Dahl (WT; n=9‐19/group) or Dahl rats lacking T cells (CD247 knock out [KO] rats; n=3‐7/group) were randomized to either a control normal fat diet (NF; 7.2% calories from fat) or a high fat diet (HF; 36% calories from saturated fat) for 10 weeks. Following treatment, a subset of livers were processed for hematoxylin and eosin staining and liver steatosis was scored. Gonadal adipose tissue was extracted and weighed.

Results

Ten weeks of HFD increased liver steatosis in WT males and females (PInteraction=0.84, PDiet=0.01, Psex=0.75, Table 1), but not in T cell deficient animals (PInteraction=0.35, PDiet=0.29, Psex=0.04). While HFD increased gonadal adipose tissue weight in both males and females, this weight was increased more in the females (PInteraction=0.006, PDiet<0.0001, Psex<0.0001). T cell deletion abolished the HFD‐induced weight gain in gonadal adipose tissue (PInteraction=0.98, PDiet=0.14, Psex=0.01).

Discussion

These data confirm the hypothesis that T cell KO prevents the HFD‐induced increases in adiposity in the liver and gonadal adipose tissue. Lipid deposits in these areas are detrimental for cardiovascular health. Lipid deposition in the liver is a strong predictor of insulin resistance. Furthermore, chronic ingestion of a HFD increases adipose tissue expansion, which promotes inflammation and oxidative stress. Since the majority of Americans are consuming large amounts of fat, investigating the mechanisms underlying HFD‐induced adiposity will help identify targets to improve cardiovascular health.
Response of Subsarcolemmal and Intermyofibrillar Mitochondria to Exercise and Doxorubicin‐Induced Cardiotoxicity
Authors: Montalvo Ryan , Boeno Franccesco P. , Ozdemir Mustafa , Dowllah Imtiaz M. , Doerr Vivian , Bomkamp Matthew P. , Nguyen Branden L. , Smuder Ashley J.
Published: May 2022

The chemotherapeutic agent doxorubicin (DOX) is highly effective at limiting cancer progression. However, systemic DOX treatment results in off‐target accumulation within cardiac mitochondria, which can cause aberrant signaling leading to dose‐dependent heart failure in cancer patients and survivors. Exercise has demonstrated preclinical and clinical efficacy in limiting the cardiotoxic effects of DOX by putatively improving mitochondrial quality and function, although this mechanism has not been fully explicated. Therefore, to further understand the molecular determinants of exercise‐mediated protection against DOX‐induced mitochondriopathy and cardiotoxicity we independently examined the cardiac subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subpopulations. Female Sprague Dawley rats were divided (n=10/group) into 1) Sedentary‐Saline; 2) Sedentary‐DOX; 3) Exercise‐Saline; and 4) Exercise‐DOX groups. Exercise groups underwent 10 days of aerobic treadmill running (~70% VO2max, 1hr/day) with 2 days of rest after day 5. Twenty‐four hours following the last exercise bout animals received saline or DOX (20mg/kg IP) treatment. Dependent measures were evaluated 48 hours post treatment. To demonstrate that the exercise preconditioning protocol was sufficient to elicit cardiorespiratory adaptations, all groups performed an exercise tolerance test (ETT). The ETT revealed a significant increase in cardiorespiratory fitness in both exercise‐trained groups, regardless of saline or DOX treatment. In addition, evaluation of the myocardial performance index supported the premise that exercise preconditioning induces a cardioprotective phenotype against DOX toxicity. Further, analysis of the cardiac SS and IMF mitochondrial subfractions indicate that exercise preconditioning protects against DOX‐induced reactive oxygen species (ROS) emission in the SS fraction. Interestingly, IMF mitochondrial ROS emission was not affected by exercise or DOX treatment. In contrast, the IMF mitochondria respiratory control ratio (RCR) was significantly elevated in Exercise‐Saline animals compared to both sedentary groups, while no differences existed in the SS mitochondria. These findings indicate that 1) exercise preconditioning is a beneficial non‐pharmacological approach to improve cardiac outcomes following DOX treatment and that 2) cardiac mitochondrial subfractions are differentially affected by exercise and DOX treatment.
The mitochondrial PE synthase Psd1 moonlights at ER subdomains to promote LD biogenesis
Authors: Friedman Jonathan , Gok Mehmet O. , Speer Natalie O. , Henne Mike
Published: May 2022

The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization and is a crucial factor regulating organelle shape and biogenesis. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that generates negative membrane curvature, has many cellular roles, including regulating mitochondrial architecture and lipidation of the autophagy factor LC3. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which canonically operates on the mitochondrial inner membrane, unexpectedly also localizes to the ER. However, it has been unclear what the role of ER‐localized Psd1 is. We now resolve this mystery and demonstrate that ER‐localized Psd1 is a critical regulator of lipid droplet (LD) biogenesis at ER subdomains. While it is constitutively targeted to the ER membrane, Psd1 transiently concentrates on the ER to LD attachment sites specifically during stimulated LD biogenesis. Using an inducible LD biogenesis assay, we demonstrate that ER‐localized Psd1 is required for normal LD formation. Further, we identify a LD binding motif on Psd1 and show that this motif is required for Psd1 to influence LD morphogenesis. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in fission yeast, though occurs through a distinct targeting mechanism than in budding yeast. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs are organized and shaped by the spatial positioning of a single PE synthesis enzyme.
The Role of TLR4 in Pentachlorophenol Stimulation of IL‐1β Production in Human Immune Cells
Authors: Seaton‐Terry Aleshia , Whalen Margaret
Published: May 2022

The environmental contaminant pentachlorophenol (PCP) is detected in human blood samples at levels as high as 5 µM. Exposure to PCP presents a serious risk to humans. PCP has been associated with respiratory diseases and cancer, showing a strong association with non‐Hodgkin’s lymphoma, multiple myeloma, and kidney cancer. Interleukin‐1 βeta (IL‐1β) is a potent pro‐inflammatory cytokine produced by immune cells. Production of IL‐1β by immune cells is normally stimulated when pathogen‐ or damage‐associated molecular patterns (PAMPS/DAMPS) activate toll‐like receptor (TLR) regulated pathways. It is well known that abnormal production of IL‐1β is responsible for chronic inflammation (inflammation in the absence of injury or infection), which is implicated in several diseases such as autoimmune diseases and cancer. Previous work has demonstrated that PCP causes human immune cells to produce elevated levels of IL‐1β and that PCP‐induced stimulation of IL‐1β production was dependent on activation of MAP kinases, which are components of TLR signaling pathways. It has not yet been established whether PCP associates with TLR. In this study, we examined whether PCP requires TLR4 to stimulate production (secreted + intracellular levels) of IL‐1β in human peripheral blood mononuclear cells (PBMC). Cells were treated for 1 h with a selective TLR4 inhibitor (TAK242), or appropriate control, prior to exposure to 5, 2.5, and 1 µM PCP. Secreted IL‐1β was measured by ELISA and intracellular IL‐1β was determined by Western blot. The results showed decreased PCP‐induced production of IL‐1β when TLR4 was blocked by the inhibitor. This suggests that PCP uses (to some extent) the TLR4 receptor to stimulate the production of IL‐1β in human immune cells. These findings provide insight into the mechanism by which PCP may lead to chronic inflammation and its associated pathologies.
How the lung folds itself: biophysical insights from evolution
Authors: Nelson Celeste M.
Published: May 2022

Evolution has generated an enormous diversity of biological form. Given this diversity, it is highly likely that every tissue structure that one can imagine has been built by the embryo of one species or another. We are interested in uncovering the physical (mechanical) mechanisms by which epithelial sheets fold themselves into branching tubes in the embryo, and using those mechanisms to engineer tissues in culture. Over the past half century, developmental biologists have identified several biochemical signaling pathways and genetic control mechanisms necessary for tissue morphogenesis. In parallel, biological systems must obey Newton’s laws of motion, and physical forces need to be generated in order to sculpt simple populations of cells into complex tissue forms. Inspired by the evolutionary diversity of embryonic forms, we have combined time‐lapse imaging analysis with computational modeling and engineered tissue models to identify the mechanical forces and downstream signaling pathways that are responsible for generating the diverse architectures of the lungs from birds, mammals, and reptiles. I will describe our recent insights into the different physical mechanisms used to fold the epithelium across these three classes of vertebrates, and possible explanations for the divergence of the avian lung.