bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of an S‐adenosylmethionine Dependent X‐Succinate Synthase Activating Enzyme
Authors: King‐Roberts Devin , Andorfer Mary , Drennan Catherine
Published: May 2022

Hydrocarbons and, unfortunately, hydrocarbon waste are ubiquitous in natural and man‐made environments. It is unsurprising, then, that many microbes are capable of metabolizing hydrocarbons for use as carbon sources. Such microbes can be responsible for biodeterioration, leading to environmental damage, but paradoxically also play a role in bioremediation. As a result, an understanding of their mechanisms of action, and the ability to reproduce those mechanisms in vitro, is highly sought‐after. One such hydrocarbon degradation mechanism kicks off with the anaerobic addition of hydrocarbons to fumarate by a class of enzymes known as X‐succinate synthases (XSSs). These enzymes use a glycyl radical cofactor to accomplish this difficult chemistry under mild conditions. This cofactor must be installed by a radical S‐adenosylmethionine (SAM) activating enzyme, which has presented a hurdle to obtaining activity in vitro. The most well‐characterized XSS, known as benzylsuccinate synthase (BSS), adds toluene to fumarate to form benzylsuccinate. Unfortunately, its corresponding activating enzyme (AE) does not solubly express in E. coli, which has limited research into the mechanism of XSS activation. To circumvent this barrier, we took a genome‐mining approach to identify homologs of BSS‐AE, yielding soluble homologs that expressed well in E. coli. Using EPR spectroscopy and LC‐MS/MS, we have demonstrated that the 4Fe4S cluster within one of these AEs is able to be reduced and that this AE is able to cleave SAM. Moreover, we have expressed this particular homolog’s corresponding XSS and found that hydroalkylation activity can be reconstituted in vitro, as observed by LC‐MS/MS. It is our hope that this system will allow for a greater understanding of XSS‐AEs and their corresponding XSSs, as well as for the development of these enzymes as synthetically useful catalysts.
Renal adaptations in gestational hypertension preserves K+ while minimizing Na+ retention
Authors: Stadt Melissa , Layton Anita
Published: May 2022

During pregnancy, major adaptations in renal morphology, hemodynamics, and transport occur to achieve the volume and electrolyte retention required in pregnancy. These changes are complex, and in isolation are sometimes counterintuitive. Additionally, during pregnancies complicated by a disorder, such as gestational hypertension, altered renal function from normal pregnancy values occur. The goal of this study is to analyze how altered renal function during gestational hypertension impacts renal function during pregnancy. We hypothesize that hypertension‐induced changes in renal transport pattern shifts Na+ transport to downstream segments, while retaining sufficient K+ for pregnancy needs. To test this hypothesis, we developed epithelial cell‐based computational models of solute and water transport in the superficial and juxtamedullary nephrons of the kidney for a pregnant rat with hypertension. The model represents reduction in proximal tubule and medullary loop transporters. As a result, urine Na+ and volume output are predicted to increase, which is evidence for pressure natriuresis. Due to differential changes in transporter activities, Na+ transport load is predicted to shift to distal segments, where transporters are upregulated. Consequently, natriuresis and diuresis are partially counteracted. Simulation results also suggest that differential regulation of medullary (decrease) and cortical (increase) thick ascending limb transporters is important to preserve K+ while minimizing Na+ retention during gestational hypertension.
Molecular and Functional Characterization of the Dual‐Specificity Phosphodiesterase Type 10A in Human White Adipocytes
Authors: Hoffmann Annett , Springer Rebecca , Knop Juna‐Lisa , Otto Christoph , Schlegel Nicolas , Hankir Mohammed
Published: May 2022

The second messenger cyclic nucleotides cAMP and cGMP are key effectors of thermogenesis in adipocytes in response to a diverse array of cues. Their intracellular levels are normally regulated by the hydrolytic action of phosphodiesterases (PDEs), a conserved superfamily of enzymes composed of 12 families in humans (PDE1‐12) with distinct substrate specificities and tissue distribution profiles. Phosphodiesterase inhibitors are thus promising drugs for the treatment of metabolic disease by promoting cyclic nucleotide‐mediated adipose tissue thermogenesis, but so far mainly those that elevate either cAMP or cGMP signaling alone have been studied. We previously showed that inhibition of the dual‐specificity phosphodiesterase type 10A (PDE10A) with the small molecule drug MP‐10 sufficiently induces a thermogenic gene expression program in differentiated human white pre‐adipocytes (HWP‐ds) to a similar degree as combined cAMP and cGMP treatment. Here, we asked (1) which PDE10A isoforms are expressed in HWP‐ds, (2) the downstream signaling pathways PDE10A controls, and (3) how PDE10A expression is influenced by inflammatory stimuli in this cell type. Immunoblot analysis revealed that both the cytosolic (PDE10A1) and membrane‐bound (PDE10A2) isoforms of PDE10A are highly expressed in HWP‐ds, predominantly in the former subcellular compartment. This was confirmed by fractionation and immunofluorescence analyses. Inhibition of PDE10A with MP‐10 dose‐dependently increased PKA and PKG signaling as revealed by phosphorylation of cyclic response element binding protein (CREB) and vasodilator‐stimulated phosphoprotein (VASP) at S133 and S239, respectively. Consistently, the stimulation of lipolysis by MP‐10 was fully prevented by combined pre‐incubation of HWP‐ds with selective PKA and PKG inhibitors. Finally, mimicking the adipose tissue microenvironment in obesity by applying pro‐inflammatory cytokines onto HWP‐ds caused an early (4‐hour) and sustained (24‐hour) increase in PDE10A mRNA expression. These results provide new insight into the molecular character, function, and regulation of PDE10A in human adipocytes and further justify the use of selective inhibitors for the treatment of metabolic disease through stimulating adipose tissue thermogenesis in a more efficient and targeted manner.
Distribution of c‐Fos Labeling Elicited by Intragastric Copper Sulfate: Comparison with Labeling Induced by Galvanic Vestibular Stimulation
Authors: Amin Pooja R. , Sampson Jacob T. , Dong Xiaoying , Bielanin John P. , Murphey Charles P. , Shulgach Jonathan A. , King Brandon L. , Douglas Nerone K. , Yates Bill J. , Balaban Carey D.
Published: May 2022

The neural pathways that produce nausea and emesis are poorly defined. Although it is widely postulated that a “final common pathway” is activated during emetic responses elicited by any stimulus, this hypothesis has not been adequately tested. In a prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014) we mapped the distribution of Fos labeling in the feline brainstem elicited by galvanic vestibular stimulation (GVS) that evoked retching and other indicators of nausea. As a comparison, in the present study we mapped Fos labeling patterns induced by intragastric administration of copper sulfate (CuSO4), which produces emesis by activating vagal afferent fibers. Emetic responses slowly build‐up during GVS, but occur rapidly following CuSO4 administration. In 10 felines (6 male, 4 female), doses of CuSO4 ranging from 83 to 322 mg were administered through an intragastric catheter or gavage, and ~2 hours later animals were perfused with paraformaldehyde. The brain was sectioned and processed for Fos labeling as in our prior study (Balaban et al., PLoS ONE 9(1): e86730, 2014). Three additional animals served as controls and did not receive intragastric CuSO4. Some brainstem areas exhibited Fos labeling following either GVS or CuSO4 treatments, as shown in Fig. 1: the commissural, medial and lateral regions of nucleus tractus solitarii, lateral reticular nucleus, medullary lateral tegmental field, subtrigeminal nucleus, medullary raphe nuclei, parabrachial nucleus, and locus coeruleus. However, the labeling patterns also diverged: CuSO4 administration produced virtually no labeling in the vestibular nuclei (which was heavy following GVS), more labeling in nucleus tractus solitarii and the medullary raphe nuclei, and less labeling in the pontine raphe nuclei. These findings support the hypothesis that some brainstem areas mediate nausea and emesis induced by any stimulus, independent of how quickly the responses develop, but also suggest that unique pathways may be activated by particular emetic stimuli.
Unusual Nervous Contribution to the Formation of the Inferior Gluteal Nerve: Clinical and Surgical Outcomes
Authors: Portela‐Vázquez María A. , Acevedo‐Arroyo Alexandra N. , La Puerta‐Resto Marizabel , Jiménez‐Dietsch Sofía , Quiñones‐Rodríguez Jailenne I.
Published: May 2022

Knowledge of anatomical variations within the gluteal region is important to accurately diagnose and avoid potential iatrogenic nerve injuries. An unusual nerve course within the gluteal region was identified. The posterior femoral cutaneous nerve (PFCN) and S3 nerve root contributed to the formation of the inferior gluteal nerve (IGN), which then continued its path to innervate the gluteus maximus muscle (GMax). Based on these findings, the IGN received fibers from spinal levels L5, S1, S2 and S3. This variation has clinical and surgical implications. Peripheral nerve anomalies are important for physicians and should be taken into consideration for some radiological diagnoses and surgical procedures such as total hip arthroplasty which is the most common surgical procedure in which the IGN can be injured.
Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage
Authors: Sojitra Mirat , Sarkar Susmita , Maghera Jasmine , Schmidt Edward N. , Rodrigues Emily , Carpenter Eric J. , Seth Shaurya , Vinals Daniel F. , Bennett Nicholas J. , Reddy Revathi , Khalil Amira , Xue Xiaochao , Bell Michael R. , Zheng Ruixiang B. , Zhang Ping , Nycholat Corwin , Bailey Justin J. , Ling Chang‐Chun , Lowary Todd L. , Paulson James C. , Macauley Matthew S. , Derda Ratmir
Published: May 2022

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate‐based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido‐modified glycans. The display of glycans on each phage virion can be controlled from 30‐1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec‐2) and DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin), and in live mice (Nat. Chem. Bio. 17, 806–816, 2021). From a mixture of 50‐100 multivalent glycan probes, LiGA identifies the glycan‐phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo.

Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec‐7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec‐1 using a cell‐based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans‐ glycan to overcome the cis‐ masking on the surface of immune cells.
Dithiolethiones D3T and ACDT Protect Against Ferroptosis Induced by Erastin and RSL3
Authors: Kulkarni Neha , Gadde Rajitha , Betharia Swati
Published: May 2022

Ferroptosis is an iron‐dependent and non‐apoptotic form of cell death characterized by increased lipid peroxidation. It is morphologically, biochemically, and genetically different from other types of cell death. Erastin and RSL3 are the canonical inducers of ferroptosis. While erastin inhibits the cystine/glutamate antiporter (xCT) leading to intracellular glutathione (GSH) depletion, RSL3 directly inhibits the glutathione peroxidase‐4 (GPX4) enzyme, resulting in increased lipid peroxidation and ferroptosis. Dithiolethiones 3H‐1,2‐dithiole‐3‐thione (D3T) and 5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester (ACDT) are lipophilic, organosulfur compounds that are known to activate the Nrf2 transcription factor and induce the cellular antioxidant machinery. They have previously been shown to be cytoprotective against various oxidative stressors both in vitro and in vivo. Furthermore, as lipophilic antioxidants are known to oppose ferroptosis and the activation of Nrf2 has been reported to inhibit this form of cell death, the present study was aimed to evaluate the protective effect of dithiolethiones against erastin‐ and RSL3‐induced ferroptosis in U‐87 MG human astrocytoma cells. Ferrostatin‐1, a known ferroptosis inhibitor was used as a standard comparator. Cells were pretreated with either 50 µM D3T, ACDT, or 10 µM ferrostatin‐1 for 24 hours followed by another 24 hour exposure to either 20 µM erastin or 10 µM RSL3. Pretreatment with dithiolethiones reversed erastin‐induced cell death measured by MTS cell viability assay and upregulated xCT transporter expression indicated by western blotting. Dithiolethiones increased intracellular GSH (GSH‐Glo luminescence assay) and alleviated lipid peroxidation (TBARS assay) against erastin. RSL3‐induced ferroptosis and lipid peroxidation were also counteracted by both the dithiolethiones, however no effect was observed on GPX4 enzyme levels. These effects of dithiolethiones were comparable to those of the standard ferroptosis inhibitor ferrostatin‐1. Collectively, our data indicate a strong ability of dithiolethiones to counteract ferroptosis, and is the first such study in astrocytes. D3T and ACDT can therefore be potential therapeutic candidates in the treatment of ferroptosis‐related brain disorders.
Proteins Interacting with S‐Nitrosoglutathione reductase to Regulate Nitric Oxide Homeostasis
Authors: Kaur Yadwinder , Treffon Patrick , Vierling Elizabeth
Published: May 2022

In plants, nitric oxide (NO) is a crucial signaling molecule involved in many physiological processes and stress pathways. NO can influence plant germination, flowering, and root development1. NO is usually found in an adduct with glutathione, forming S‐nitrosoglutathione (GSNO), which can affect protein S‐nitrosation, an important post‐translational modification that can alter protein activity and stability. The enzyme S‐nitrosoglutathione reductase (GSNOR) catalyzes the irreversible NADH‐dependent reduction of GSNO. GSNOR’s ability to regulate GSNO levels and subsequently affect S‐nitrosation activity makes it a critical enzyme in NO signaling2. The objective of this project is to identify proteins that interact with GSNOR to regulate its activity and stability, thereby impacting NO homeostasis. Based on previous experiments, we hypothesize that thioredoxins and catalase may be recovered as interacting candidates. We are utilizing enzyme‐catalyzed proximity labeling with the biotin ligase, TurboID, coupled to mass spectrometry to identify interacting partners of GSNOR in the plant model Arabidopsis thaliana. Currently, we have shown that a GSNOR‐TurboID fusion protein that has been transformed into a GSNOR null mutant can rescue the mutant phenotype without displaying any adverse effects on plant growth. As controls, A. thaliana lines carrying TurboID fused to YFP and targeted to the nucleus or cytosol have also been characterized. Importantly, upon treatment with biotin, TurboID produces strong biotinylation signals in these lines. These findings indicate that TurboID is suitable for use in A. thalianaand that biotinylated proteins can be recovered for identification by mass spectrometry. These experiments will advance the current understanding of GSNOR’s regulation, leading to an improved model of NO signaling.

1. Xu, S., D. Guerra, U. Lee, & E. Vierling. S‐Nitrosoglutathione reductases are low‐copy number, cysteine‐rich proteins in plants that control multiple developmental and defense responses in Arabidopsis. Front. Plant Sci. 4: 1‐13 (2013). doi: 10.3389/fpls.2013.00430. 2. Lee, U., Wie, C., Fernandez, B. O., Feelisch, M. & Vierling, E. Modulation of nitrosative stress by S‐nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell. 20: 786–802 (2008). doi: 10.1105/tpc/107.052647.
Phenolic Extract(s) from Wild Blueberries promote(s) wound healing via vascularization
Authors: Adekeye Tolu E. , VandenAkker Natalie , Tsakiroglou Panagiotis , Weber James , Klimis‐Zacas Dorothy
Published: May 2022

Wounds occur in over 15 million people with approximately $60 billion spent annually on wound care. Deficient vascularization is a significant factor that promotes chronic and non‐healing wounds, ultimately resulting in amputation. Currently, most clinical wound treatments are antibiotics for infection control. Therefore, chronic wounds such as diabetic foot ulcers, ischemic limbs, and pressure ulcers lack treatments for healing. Our lab reported that phenolic extracts (PE) from wild blueberries (WB) promote angiogenesis in HUVEC cells via cell migration, angiogenesis, and VEGF/P13K/AKT pathway. However, the in vivoeffect is still unknown. To investigate the in vivo pathways of PE from WB on vascularized wound healing, we extracted PE from WB by Folin‐Ciocalteu method, and incorporated them into a serum [hyaluronic (HA) gel] and a cream carrier. We grouped fifty‐six Sprague‐Dawley rats into seven groups as follows: Group1 as Control (nothing applied on the skin); Group 2 as the cream base, without the PE; Group 3 as the HA gel base, without PE; Group 4 as the HA gel base with 500 μg/ml of the PE; Group 5 as the cream base with 500 μg/ml of PE; Group 6 as the HA gel base with PE at 1000 μg/ml; and Group7 as the cream base with PE at 1000 μg/ml. We created dorsal surgical wounds on each rat and treated them accordingly for six days. Photographs were taken daily to assess wound closure. Percentage wound closure was determined using ImageJ. Thereafter, the tissues were excised and stained with H&E, visualized and quantified under light microscope for proliferating cells associated with vascular wound closure. We observed 12% increase in wound closure and significant increase in endothelial cells on the wound area with 500 μg/ml of PE in serum (HA gel) treatment when compared to the control without treatment. The results support the potential effect of PE to promote vascularized wound healing. Frozen tissues are currently being analyzed for genes and proteins associated with vascularization. This project is innovative because it provides new insights into the metabolomic pathways of PE treatment in rats used as a wound healing model. Results of this project may be beneficial to patients with chronic wounds promoting vascularization and wound healing, with all natural, inexpensive, no side effect wound healing products, using Phenolic extracted from WB.
Decellularized Three‐Dimensional Biomimetic Scaffolds as In Vitro Model For Tendon And Meniscus Regeneration
Authors: Sormani Jordana M. , Dognani Melissa I. , Vetucci Viviann R. , de Souza Faloni Ana Paula , Trovatti Eliane , Amaral André C.
Published: May 2022

Traumatic and degenerative injuries to the meniscus and tendon still pose challenges to treatments available in the fields of orthopedics and sports medicine. In search of more effective therapeutic procedures based on biological strategy, regenerative medicine advances like medical science carrying‐out preclinical and clinical research necessary for establishing therapeutic efficacy and safety. This research aimed to establish the new concept of three‐dimensional decellularized biomimetic scaffolds as in vitro models to investigate the tendon and meniscus regenerative medicine approaches. For this purpose, the menisci of the swine's knee and the tendons of the deep flexor muscles of the bovine's fingers were resected, cleaned, fragmented and frozen (‐20°C) for 48 hours. After this, the fragments were thawed and decellularized by immersion in sodium dodecyl sulfate (0.5%) and Triton‐X100 (1%) solution for 72 hours. To faithfully simulate a degenerative injury, 0.05 mL of collagenase type‐1 (1mg.mL‐1/37°C/1hour) was injected into the central region of the meniscal samples. To simulate the traumatic injury, incisions were made on the surface of the samples with a scalpel. The fragments were washed in running water (3 days), immersed in formaldehyde (4%), washed again, stored in ethanol (70%), frozen (‐30°C), and submitted to the lyophilization process or embedding in paraffin for histological procedures. For microscopic characterization of tissue and injury sites, histological sections were stained with Hematoxylin and Eosin (HE) or Picrosirius and analyzed under a light microscope with a polarization filter. The results, based on qualitative analysis, demonstrate the preservation of macroscopic and microscopic architecture of the scaffold matrix, the efficacy of the decellularization procedure, and the accuracy in creating the traumatic and degenerative sites of injury. These results allow us to conclude the success of this research in establishing the concept of decellularized three‐dimensional scaffolds as a new biomimetic models applicable for in vitro assays evolving regeneration of traumatic and degenerative injuries of tendons and menisci, contributing to the advancement of preclinical research on regenerative medicine approaches.
Anti‐hypertensive effect of BPM4 on spontaneously hypertensive rats that antagonizes Dopamine βeta hydroxylase
Authors: Saini Manisha , Dey Sanjay K. , Kundu Suman
Published: May 2022

Background

Hypertension, which is described as chronically increased blood pressure above 140/90 mmHg, has emerged as a starring cause of mortality and morbidity worldwide. It is a leading risk factor for major cardiovascular events (such as heart attack and stroke), chronic kidney disease, heart failure, cognitive impairment and dementia. The dire consequences of uncontrolled hypertension indicate that controlling it will have a direct preventive effect on the vital organs. Despite the availability of a large number of classical anti‐hypertensive drugs it still affects humans adversely and poses a global disease burden thus necessitating new drugs. A non‐conventional target whose inhibition can regulate hypertension is Dopamine βeta hydroxylase (DBH). This enzyme, which plays a significant role in the catecholamine biosynthetic pathway, utilizes the body’s blood pressure (BP) lowering mechanism and thus can combat resistant hypertension.

Methodology

In the study, we have screened thousands of small molecules against the active site of DBH (PDB ID:4ZEL). Hits identified in silico were assessed for their inhibitory potential and strength of binding (Kd) in vitro. The ex vivo toxicity parameters were evaluated to get rid of toxic molecules before going for in vivostudies. Selected leads were screened in spontaneously hypertensive rats (SHRs) to evaluate their efficacy in reducing blood pressure as well as their ability to improve other cardiac parameters. Thereafter, acute as well as genotoxicity study was performed to minimize the rate of attrition in later phases of clinical development.

Results

Our best lead molecule “BPM4” inhibited DBH efficiently with an IC50 value of 18 μM and dissociation constant of 22 nM. BPM4 when administered through IP at a dose of 10 mg/kg BW significantly reduced BP with a p‐value of 0.001 as compared to the untreated group. Additionally, a significant reduction was observed in IVSs, IVSd, LVPWs and LVPWd, whereas increment was observed in internal diameter in the case of end systole and diastole (LVIDs and LVIDd). A high oral lethal dose (LD50) > 2000 mg/kg was observed in the acute toxicity test. Ames test showed no positive mutagenic effect +‐S9. No statistically significant decrease in % mitotic index nor any aberrant cells were observed in lymphocytes and bone marrow of rodents.

Conclusion

BPM4 (at 10 mg/kg BW) statistically reduced the BP as well as improved the left ventricular hypertrophy parameters in SHRS. The area of fibrosis was also decreased post treatment (cardiac fibrosis disrupts the normal myocardial structure and has a direct impact on heart function and survival). Subsequently, the compound successfully passed the three battery genotoxicity tests as it did not cause any structural damage to DNA nor did it induce any ploidy changes, holds scope for being a potent anti‐hypertensive drug candidate.
Molecular, Biochemical and Physiological Studies on the Potential Influence of the Beta vulgaris Extract to Mitigate the Effect of Chemical Colorants of Food on Male Rats
Authors: Okab Aly B. , Elbanna Sabah G. , Khalil Khaled A. Z.
Published: May 2022

Synthetic food colorings as carmoisine (E122) contains the azo group (–N=N) that linked to the aromatic rings in the chemical molecular structures used as red color. Red beetroot (Beta vulgaris) ‐ a natural antioxidant‐ is a rich plant source of nutrients that give tremendous health benefits, red beetroot includes anthocyanins and betalains, which are natural antioxidants and can protect against age‐related diseases. The objective of this study was to investigate the potential protective effects of Beta vulgaris extracts against Carmoisine induced oxidative damage among male rats. In this study animals were divided randomly into 6 groups (18 animals each). Hemoglobin significantly increased after 15 days in Red beetroot 10 mg/kg (B), Carmoisine low dose 50 mg/kg (CLD), and beetroot + carmoisine high dose 10 mg/kg + 100 mg/kg (BCHD), while decreased after 30 days in CHD and increased after 45 days in CHD, BCLD, and BCHD. While WBCs increased after 15 and 45 days but decreased after 30 days in all groups. Thiobarbituric acid reactive substances (TBARS) in blood plasma increased after 15 days in all groups, while, after 30 days, TBARS in blood plasma decrease in B, CLD, BCHD, whereas, TBARS in liver increased in all groups, and TBARS in testes increased in all groups except B. After 45 days TBARS in testes decreased in B, increased in CLD, CHD, and BCHD. Cytochrome b5 in liver increased after 15 days in all groups, except BCLD. Cytochrome P450 increased in all groups, while, after 30 days cytochrome b5 and cytochrome P450 decreased in BCLD. After 45 days cytochrome b5 increased in CHD, and decreased in BCLD. While, cytochrome P450 increased in CLD, CHD, and BCHD, and decreased in BCLD. Superoxide dismutase (SOD), catalase in liver male rats after 15 days increased in all groups, while DNA breakage decreased in BCLD and BCHD, whereas, after 30 days catalase decreased in CLD and BCHD, and DNA breakage increased in CLD and decreased in BCHD, however, after 45 days SOD decreased in BCHD, catalase decreased in BCLD and BCHD, however DNA breakage increased in all groups except B. Catalase in testes of male rats after 15 days decreased in all groups except BCHD, however, testes DNA breakage decreased in B and CHD, while, after 30 days SOD increased in all groups, while catalase decreased in all groups except CHD, while, DNA breakage increased in CHD, but decreased in BCLD and BCHD, whereas, after 45 days catalase increased in all groups, DNA breakage decreased in B, BCLD, and BCHD, except, CLD and CHD.

From the present results it was concluded that Co‐administration of beetroot decreased the oxidative stress in liver and testes and also prevent liver damage, also decreased DNA breakage.
Optimization of a Chaperone Protein Disaggregase Complex for Neurodegenerative Disease
Authors: Qiang Evan , Gorenberg Erica , Chuang Eddie , Braun Gabriel , Shorter James
Published: May 2022

Toxic and pathologic protein aggregation is a common feature of neurodegenerative diseases from Alzheimer’s and Parkinson’s diseases to amyotrophic lateral sclerosis (ALS). Chaperone proteins such as the metazoan heat shock protein 70 (Hsp70), Hsp40, and Hsp110 complex can identify these misfolded aggregates and restore them to their functional native states. However, previous studies have used a wide range of chaperone complex components and proportions. Using aggregated firefly luciferase as a model substrate, we set out to optimize the stoichiometric ratio for the disaggregation of these aggregates in vitro. We tested ratios of Hsp70:40:110 from 1:10:1 to 1000:100:1 and used the luminescence of firefly luciferin as a measure of disaggregation activity. With this data, we proceeded to refine the canonical Hsp70/40/110 disaggregase network against fused in sarcoma (FUS), a protein that forms pathologic protein aggregates in ALS and frontotemporal dementia (FTD). We used absorbance measurements at 395 nm from a high‐throughput plate reader to determine the turbidity levels of FUS aggregates with various combinations of Hsp70/40/110. Together, our findings support the existence of an ideal Hsp70/40/110 system which provides a framework to optimize the disassembly of protein aggregates in other neurodegenerative diseases.
Hepatoprotective Effects of Astragaloside IV and Formononetin Against Oxidative Injury
Authors: Tran Quoc‐Anh , Duong Nhi , Luu Vy T. , Xiong Hou M. , Ho Linh
Published: May 2022

The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST‐IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha.

We first tested whether AST‐IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST‐IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST‐IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis.

In addition, we assessed rescue effects of AST‐IV and formononetin on t‐BHP‐induced oxidative injury in AML12 hepatocytes under three separate conditions: pre‐treatment with AST‐IV or formononetin, post‐treatment with AST‐IV or formononetin, and both pre‐treatment and post‐treatment with AST‐IV and formononetin. Through CCK‐8 analysis, we identified that both pre‐treatment and post‐treatment significantly increased cell proliferation on induced‐oxidative stress AML12 cells compared to no treatment and control. Pre‐treatment of AML12 cells before induction of oxidative stress with AST‐IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre‐treatment and post‐treatment with AST‐IV or formononetin. Post‐treatment AST‐IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non‐treatment and control. Significantly AST‐IV and formononetin showed induction of the SIRT3 protein on tert‐butyl hydroperoxide (t‐BHP) induced oxidative stressed AML12 hepatocytes by WB.

Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST‐IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST‐IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein‐linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes.
Identification of protein markers for skeletal muscle‐derived extracellular vesicles (SkM‐EVs) by quantitative proteomics reveals how SkM‐EVs function in vivo
Authors: Watanabe Sho , Sudo Yuri , Sakurai Hidetoshi , Sato Ryuichiro , Yamauchi Yoshio
Published: May 2022

Background

Skeletal muscle serves as a secretory organ; it releases various soluble factors called myokine. They act as mediators for cell‐cell communications in autocrine, paracrine, and endocrine fashions. Extracellular vesicles (EVs) have been proposed to be a partial paracrine and/or endocrine effectors by facilitating the exchange of biological cargoes among cells and tissue. Some in vitro study showed that skeletal muscle released EVs and skeletal muscle‐derived EVs (SkM‐EVs) could induce biological effects in recipient cells. However, whether the skeletal muscle actively releases EVs in vivo, how much proportion of plasma EVs are derived from this tissue, and where SkM‐EVs are delivered and exert their roles remain largely unknown. Here, we aimed to identify SkM‐EV marker proteins by quantitative proteomics on human and mouse myocyte‐derived EVs to evaluate SkM‐EVs abundance in vivo. In addition, we explored whether SkM‐EVs isolated from mice could exert biological functions.

Methods

Proteomic analysis of EVs released from mouse C2C12 myotubes and human iPS‐derived myocytes was performed to identify potential SkM‐EVs markers. The levels of SkM‐EVs in plasma and muscle interstitium were evaluated by the abundance of identified SkM‐EVs protein markers. Based on previous methods, we developed a modified protocol to isolated SkM‐EVs from muscle interstitium. We confirmed that muscle interstitium‐EVs were incorporated into C2C12 myoblast and investigated the expression levels of myogenesis‐related genes.

Results& Discussion

We first determined proteomic profiles of EVs released from C2C12 myoblasts, C2C12 myotubes, and hiPSC‐derived myocytes and identified several proteins that serve as potential markers for SkM‐EVs. Among them, we identified ATP2A1, β‐enolase, and desmin as reliable SkM‐EV marker proteins. Using these protein markers, we confirmed that SkM‐EVs were present in muscle interstitium but little in the blood, which were supported by our electron microscopy. Unexpectedly, the abundance of SkM‐EVs present in plasma and muscle interstitium was not changed in response to acute exercise. Furthermore, we showed that SkM‐EVs accumulated in muscle interstitium space were incorporated into C2C12 myoblast and promoted muscle differentiation by regulating gene expression of Pax7 and myosin heavy chain at least partially through myomiRs (miRs‐1, ‐206, ‐431, and ‐486). We expect that molecular characterization and in vivo distribution of SkM‐EVs could provide a conceptually new basis how skeletal muscle EVs exert their functions.
The Effect of Glycosylation Modulators on the Trafficking and Interaction of Spike Protein S1 Subunit and Angiotensin‐Converting Enzyme 2
Authors: El Khoury Marianne , Wanes Dalanda , Hoter Abdullah , Naim Hassan Y.
Published: May 2022

The betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing worldwide pandemic. The spike (S) glycoprotein of SARS‐CoV‐2 is a heavily glycosylated trimer that binds to host cell receptor angiotensin‐converting enzyme 2 (ACE2), initiating viral entry. Evidence also shows that the S glycoprotein interacts with other receptors including dipeptidyl peptidase IV (DPPIV). Glycosylation is a post‐translational modification important for proper folding and trafficking of membrane and secretory proteins. It plays a critical role not only for host cell proteins, but also in viral virulence, immunity evasion and receptor binding capabilities. This study focused on the modulation of glycosylation and its effects on the trafficking of the S1 subunit, ACE2 and DPPIV, in addition to its effect on their interactions. For this purpose, the S1 subunit was expressed in COS‐1 cells in the presence or absence of N‐butyldeoxynojirimycin (NB‐DNJ), an inhibitor of the ER‐located a‐glucosidases I and II, 1‐deoxymannojirimycin (dMM), an inhibitor of Golgi‐mannosidase I, or benzyl‐N‐acetyl‐α‐galactosaminide (benzyl‐GalNac), an inhibitor of O‐glycosylation. The data demonstrate that NB‐DNJ affects substantially the trafficking and secretion of S1 protein, while its overall synthetic levels remained unchanged. A similar effect was observed upon treatment of the cells with the benzyl‐GalNac. On the contrary, the presence of dMM in the culture medium did not affect the secretion of S1. Together, the data suggest that folding events implicating calnexin in the ER, the site of action of NB‐DNJ, as well as O‐glycans that are modulated by benzyl‐GalNac are crucial for the secretion of the S1 protein. We further investigated the trafficking of ACE2 and DPPIV in lung Calu‐3 and intestinal Caco‐2 cells in the presence or absence of the glycosylation modulators and their interaction with S1. The data demonstrate a reduced binding capacity of S1 to ACE2 in the presence of NB‐DNJ and dMM, while this interaction was enhanced in Calu‐3 cells upon inhibition of O‐glycosylation by benzyl‐GalNac. We conclude that glycosylation modulators differentially act on the secretory pathway of S1 and the receptors. By virtue of its negative impact on both the secretion of S1 as well as interaction with its receptor ACE2, NB‐DNJ may be considered as a potential therapeutic drug that may act at the level of viral entry and exit.
Type‐2 Diabetic/Obese Patients with Associated Vascular Disease Have Increased Circulating Acetylated HMGB1
Authors: Liu Zeyu , Raviprakash Srishti , Williamson Kelly , Mota Roberto I.
Published: May 2022

Introduction

Type‐2 Diabetes (T2D) is highly associated with peripheral artery disease (PAD), with both PAD and T2D highly associated with chronic inflammation. High mobility group box 1 (HMGB1) is key in regulating both inflammation and diabetes. Acetylated HMGB1 is the extracellularly released form of HMGB1 from post translational modifications (PTM) that occur after nuclear HMGB1 turnover. Acetylated HMGB1 has significant cytokine/chemokine activity. Plasma HMGB1 high molecular weight (HMW) complexes have been identified in the literature as PTM in chronic inflammation. Glycoprotein cytokine Osteoprotegerin (OPG) is a known marker for vascular disease, with research showing that high levels of OPG correlate tightly with increased levels of HMGB1 in T2D patients with PAD. We hypothesize that obese, T2D patients will display elevated levels of circulating acetylated HMGB1 in HMW complexes and that we will find associated high circulating OPG in same patients.

Methods

Plasma was collected from 25 male/female adult patients diagnosed with chronic T2D sorted by sex, obese or lean, w/ and w/o vascular disease. Healthy and trauma patients plasma was used as experimental controls. Diluted plasma (1:25) were denatured in SDS page gels and ran for immunoblots with luminescence using the primary antibodies HMGB1 (abcam ab18256) and acetyl‐HMGB1 (Aviva OASG03545). OPG quantification was measured in same plasma samples as above by ELISA (Thermofisher TNFRSF11B) following the manufacturer protocol. Data is represented as mean±SEM. A total of 5‐10 samples per experimental group were analyzed, representative images of immunoblots are shown. Statistical analysis was performed using t‐test and ANOVA with Tukey’s correction.

Results

Immunoblot analysis showed that there is an increase in plasma HMGB1 in plasma samples from T2D, obese patients with vascular disease; predominantly acetylated HMGB1 in HMW complexes (>100kDa) were present within obese‐T2D patients compared to both healthy patients and trauma patients. Since we wanted to understand if vascular disease was associated in plasma samples from these patients, we performed OPG quantification. When we analyzed our cohort, results showed increased OPG in obsese‐T2D patients compared to lean T2D patients with a clear difference by sex (males>females). Obese T2D males with arterial injury have significantly higher circulating OPG compared to lean T2D males with arterial injury.

Conclusions

The drastically higher HMW complexes found uniquely in obese/T2D patients identifies acetylated HMGB1 as the predominant circulating form of HMGB1 in obese/T2D patients. Findings show there might be some association of elevated circulating OPG and acetylated HMGB1 as potential biomarkers. Future studies will focus on understanding the role of acetyl‐HMGB1 in the disease progression of T2D/obese patients with vascular disease.
Enhanced Gold Nanoparticle aggregation with cancer cell DNA for improved radiation therapy and immune response in cancer treatment
Authors: Frontz Amye , Demp Jay , Miller Keith R.
Published: May 2022

In the United States, cancer is the second leading cause of death after heart disease and hundreds of thousands of people pass away each year due to this deadly disease. Many treatments for cancer are nonselective and do not provide long‐lasting immunity in case of recurrence. Gold nanoparticles can provide a more targeted radiation therapy and better effectiveness through heat generation and formation of reactive oxygen species via hydrolysis reaction which can cause DNA damage in the tumor. Unfortunately, gold particles have low photothermal transduction efficiency and must be aggregated to improve performance. To achieve this, positively charged gold nanoparticles were aggregated with MDA MB 231 cancer cell DNA to increase gold nanoparticle aggregation, to elicit higher photothermal efficiency, and to stimulate an immune response. In vitro analysis demonstrated DNA size and concentration dependent gold nanoparticle aggregation, which varied little in the pH range of 5.5 to 8.6. Furthermore, these aggregates elicited an immune response via delivery of DNA for direct transfection of antigen presenting cells. Tumor necrosis factor alpha released from mouse RAW macrophages increased in the presence of the cancer cell DNA aggregated gold particles. This work suggests that the promise of combination therapy using cancer cell DNA not only can improve photothermal therapy of gold nanoparticles, but also can elicit an immune response in cancer treatment.
Investigating the Contributions of Human Body Donors in the United States
Authors: Wyatt Taylor B. , Bagian Lauren K. , Balta Joy Y.
Published: May 2022

Working with human cadavers for anatomical education dates back to the Middle Ages. In modern day, anatomist still believe the best way to learn anatomy is through the wonderful gift of human body donors (Balta et al, 2015). Additionally, teaching the general public anatomy may increase health literacy, allowing individuals to make healthier decisions for their body (Taylor et al, 2017). In this study we aim to investigate the work supported by human body donors at academic institutions in the United States and how institutions are working to best maximize this gift. A questionnaire using Qualtrics was sent to 125 body donation programs in the United States. The questionnaire consisted of a series of conditional questions to assess how each institution works with their body donors. From the 125 invited programs, 46 have completed the questionnaire. 96.7% of the institutions utilize a hard fixation method and of those body donors undergoing hard fixation, 87.9% of the body donors support anatomical teaching. Additionally, 32.6% of the institutions have a soft preservation method. 49.5% of these donors support clinical skills training. Lastly, 45.7% of the institutions work with fresh frozen donors. 66.5% of fresh frozen body donors support clinical skills training as well. The data also shows that at these institutions, donors that have underwent the hard fixation process can be worked with indefinitely depending on their utilization and proper maintenance. Whereas soft preservative and fresh frozen body donors begin decomposition much sooner. Overall, the data suggests that hard fixation is more appropriately correlated with teaching human anatomy, while fresh frozen and soft preservation tend to support clinical skills training. This is most likely due to the sheer number of students that are taught on human body donors daily. Therefore, teaching anatomy using the human body donors, you need to utilize an embalming method that will last an extended period and is relatively easy to produce. Additionally, 92.5% of the surveyed institutions, allow faculty and staff to take photographs of body donors and 14% allow students to take photos as well for educational purposes. Lastly, 39% of the institutions allow individuals to work with human body donors for educational purposes to teach the public in outreach and continuing education settings. These unique tools and applications institutions employ allow them to further maximize their work with body donors. Also, with the appropriate embalming method, educators can teach students in an efficient and effective manner. Knowing the appropriate preservation method to utilize for a particular purpose will also help maximize the gift of donation while enhancing the education process for students and teachers.
Roles of Nav1.9 in inflammatory Mediator‐induced Activation of Vagal C‐fibers Innervating Mouse Lungs
Authors: Kim Joyce S. , Sun Hui , Meeker Sonya , Undem Bradley J.
Published: May 2022

We used NaV1.9 knockout (KO) mice to evaluate the role of NaV1.9 in chemical activation of vagal C‐fiber terminals in the mouse lungs. Single‐cell rt‐PCR of lung‐labeled neurons revealed that the vast majority of TRPV1 expressing vagal sensory neurons innervating the lungs express NaV1.9 mRNA. The mouse lungs were isolated with the vagus nerves and vagal ganglia intact. Action potentials (APs) were recorded using extracellular electrodes positioned near the relevant cell bodies in the vagal ganglion. The stimuli were applied to the receptive field via perfusion through the mouse trachea. The PAR1 agonist, TFLLR(10mM) evoked 270 ± 62 APs in wild type mice (n=23), but only 85 ± 38 action potentials in NaV1.9 ‐/‐ mice (P< 0.01, n=29). PAR1 is a GPCR stimulus, so we next evaluated a,b‐methylene ATP (1ml, 1µM) that stimulates nodose C‐fibers in the mouse lung via activation of the ionotropic P2X2/3 receptor. a,b‐Methylene ATP evoked 93 ± 18 APs in wildtype mice (n=33), compared to only 13 ± 4 APs the NaV1.9 ‐/‐ mice (P=0.0001, n= 35). The C‐fibers of both WT and NaV1.9 ‐/‐ were equally activated by rapid punctate mechanical stimulation of the receptive field with Von Frey fibers. At the patched clamped cell soma, there was no difference in active or passive electrophysiological properties of nodose neurons isolated from WT versus KO animals; rheobases were also the same with AP threshold averaging 0.34 ± 0.1 and 0.29 ± 0.06 mV/ms, respectively. However, bath‐applied ATP was more effective in evoking action potentials in neurons isolated from WT (40 % responding) vs. KO (0 % responding). The data reveal that NaV1.9 is important in the activation of nodose C‐fiber terminals evoked by inflammatory mediators that act via GPCRs or ionotropic receptors but less critical in the activation by rapid punctate mechanical or electrical stimulation. NaV1.9 provides an intriguing target for treating symptoms caused by inflammatory mediator‐induced C‐fiber activation in the airways, e.g. chronic coughing and excessive reflex bronchospasm and secretions associated with inflammatory airway disease.