bluuebunny/crossref_metadata_2025 huggingface dataset:
Extending the Catalytic HECT Domain Boundaries for the HECT E3 Ubiquitin Ligase HACE1 Increases Solubility and Enzymatic Activity
Authors: Kane Emma I. , Rich Kayla , Beasley Steven , Spratt Donald E.
Published: May 2022

There are 28 unique members of the homologous to E6AP C‐terminus (HECT) E3 ubiquitin ligase family responsible for catalytically transferring a ubiquitin moiety onto a specific substrate. This family contains a relatively conserved C­‐terminal HECT domain that mediates its ubiquitylation activity with a highly conserved cysteine residue. Of these, HECT Domain and Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1 (HACE1) is one of the ‘other’ HECT E3 ubiquitin ligases containing an N‐terminal ankyrin repeat domain responsible for facilitating substrate recognition and binding. Isolation of a biochemically pure HECT domain has proven difficult due to chronic insolubility and/or inhibited enzymatic activity. Recent in‐silico bioinformatic analyses coupled with secondary structure prediction software indicated a conserved amphipathic α‐helix preceding the N‐termini in all HECT E3 ubiquitin ligases. This α ‐helix is proposed to bind to a hydrophobic pocket found within the HECT domain. Building on recently published structural studies and in‐house biochemical and biophysical experiments, we show a drastic increase in protein yield, solubility, stability, and activity of the HECT domain of HACE1. This finding also holds true for other members of the HECT E3 ubiquitin ligase family. Our cumulative findings support a call to redefine the boundaries of the HECT domain to include this N‐terminal extension that will likely be critical for future biochemical, structural, and therapeutic studies on the HECT E3 ubiquitin ligase family.
Aging Impairs Phrenic Long‐Term Facilitation in Rats by an Adenosine‐Dependent Mechanism
Authors: Marciante Alexandria B. , Mitchell Gordon S.
Published: May 2022

Phrenic long‐term facilitation (pLTF) is a form of respiratory plasticity elicited by acute intermittent hypoxia (AIH). AIH activates competing intracellular signaling cascades, initiated by phrenic motor neuron serotonin 2 and adenosine 2A (A2A) receptors, respectively. With moderate AIH (mAIH), the serotonin‐dependent Q pathway to phrenic motor facilitation dominates, with an adenosine constraint. Consequently, spinal A2Areceptor inhibition enhances mAIH‐induced pLTF. In middle‐aged male, and geriatric acyclic female rats, pLTF is attenuated through unknown mechanisms. We hypothesize that basal spinal adenosine levels are elevated in aged rats, thereby undermining mAIH‐induced pLTF. Thus, we predict that spinal A2A receptor inhibition in aged male rats will restore pLTF to levels equal to young male rats with A2A receptor inhibition. A selective A2A receptor antagonist (MSX‐3; 10 uM, 12 uL) or vehicle (0.9% sterile saline) were delivered intrathecally (C4) in anesthetized, vagotomized, paralyzed and ventilated young (3‐6 months) and aged (20‐22 months) male Sprague‐Dawley rats prior to mAIH (3, 5 min hypoxic episodes; arterial Po2= 45‐55 mmHg). In young males, mAIH elicits ~ 60% pLTF at 60 min post‐AIH. Spinal A2A receptor inhibition significantly enhanced mAIH‐induced pLTF in young males (119 + 5%; n = 6) vs vehicle controls (55 + 9%; n = 6; p < 0.001) and time controls (12 + 4%; n = 6; p < 0.001). In aged males, A2A receptor inhibition enhanced mAIH‐induced pLTF to a greater extent (186 + 19%; n = 7) vs vehicle controls (25 + 11%; n = 7; p < 0.001) and time controls (3 + 5%; n = 4; p < 0.001). Measured basal adenosine levels in ventral C3‐C5 homogenates were significantly elevated in aged vs young males (p = 0.008). Preliminary data suggest aged female rats (16‐18 months) also have enhanced mAIH‐induced pLTF with A2A receptor inhibition (173+ 24%; n = 3) vs vehicle controls (53 + 7%; n = 3) and time controls (5 + 17%; n = 2) in persistent estrus. These findings advance our understanding of mechanisms whereby age undermines mAIH‐induced phrenic motor plasticity. Understanding age and sex effects on AIH‐induced respiratory motor plasticity is crucial as we harness therapeutic AIH to treat severe neuromuscular disorders that compromise breathing.
Dynamic Interaction Network Involving the Conserved Intrinsically Disordered Regions in Human eIF5
Authors: Marintchev Assen , Paul Eleanor E. , Lin Kai Y. , Tsai Amy W‐L , Yang Yu , Gamble Nathan , Doran Matthew
Published: May 2022

Translation initiation in eukaryotes requires multiple eukaryotic translation initiation factors (eIFs) and involves continuous remodeling of the ribosomal preinitiation complex (PIC). The GTPase eIF2 brings the initiator Met‐tRNAi to the PIC. Upon start codon selection and GTP hydrolysis promoted by its GTPase‐activating protein (GAP) eIF5, eIF2‐GDP is released in complex with eIF5. It is not known how eIF5 dissociates from its other binding partners to leave the PIC with only eIF2. Here, we report that two intrinsically disordered regions (IDRs) in eIF5, the DWEAR motif and the C‐terminal tail (CTT) dynamically contact the folded C‐terminal domain (CTD) and compete with each other. The eIF5‐CTD•CTT interaction shows modest synergy with eIF2β binding to eIF5‐CTD, whereas the eIF5‐CTD•DWEAR interaction favors eIF1A binding, instead. These findings allowed us to propose a model explaining how the rearrangement of the eIF5 intramolecular contacts can mediate remodeling of multiple interactions upon start codon selection. Using phosphomimetic mutations, we show that phosphorylation of eIF5 by Casein Kinase 2 (CK2) increases the affinity of eIF5 for eIF2β. Our results help elucidate the molecular mechanisms of stimulation of protein synthesis and cell proliferation by CK2.
Study of Trm7 Interactions With Binding Partners Trm732 and Trm734 for 2’‐O‐Methylation of the tRNA Anticodon Loop in Yeast
Authors: Rae Kellyn , Funk Holly , Guy Adrian , Guy Michael
Published: May 2022

Transfer RNAs undergo post‐transcriptional modifications, including many which occur at or near the anticodon loop and are necessary for protein synthesis. In Saccharomyces cerevisiae, Trm7 is required for 2’‐O‐methylation of tRNA and requires interaction with Trm732 and Trm734 for modifications at positions 32 and 34, respectively. These proteins are widely conserved in eukaryotes, and mutations in the human ortholog of TRM7, FTSJ1, can cause non‐syndromic X‐linked intellectual disability by decreasing or eliminating methylation activity. Through a genetic assay, we have found Trm7 residues required for interaction with Trm732 or Trm734 and the subsequent methylation of tRNAPhe. These spot test assays showed that the Trm7 Y138R variant abrogates interaction with Trm734 and the Trm7 I144A variant abrogates interaction with Trm732. To verify that these genetic results are due to impaired interaction of Trm7 variants with its binding partners, we are performing immunoprecipitation assays. In addition, we have begun to study interactions of the individual proteins and complexes with tRNAPhe through biochemical methods such as electrophoretic mobility shift assays, to determine the role of each protein in tRNA binding. Through this study of Trm7 variants, we can better understand the interaction of Trm7 with Trm734 and Trm732, which will facilitate the study of conserved homologs in humans.
Effects on Endothelin‐1 on Irisin Production By Myocytes Derived From C2C12 Cells
Authors: Valala João V. Z. , Achcar Jorge A. , Montrezor Luís H.
Published: May 2022

Skeletal muscle is one of the most metabolically active tissues. Consequently, it is highly susceptible to hormonal variations, whether by endocrine, paracrine, or autocrine pathways. This muscle is recognized as an endocrine organ that releases myokines, including irisin. Irisin acts as a signal that induces energy consumption, communicating directly with the adipose tissue, modulating the metabolic profile and increasing energy expenditure. Endothelin‐1 (ET‐1), a peptide with pro‐inflammatory activity, is a potent vasoconstrictor that interacts with receptors on the vascular smooth muscle membrane, stimulating its contraction. However, the functional relationship between ET‐1 and skeletal muscle remains unclear. The objective of this work was to study the effects of ET‐1 on cell count, cell viability, and irisin production in C2C12 cell culture. The cells were cultured for 3 and 5 days, at an initial density (day 0) of 1 x 103 cells/well (48‐well culture plates), in 200 µL of culture medium containing equine serum, antibiotics, and antifungal, in the absence of ET‐1and in the presence of ET‐1 at two concentrations (10‐10 M and 10‐6 M). The assays were performed in triplicates, with exchange of the medium every 48 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter. The MTT reduction assay was used to determine cell viability. Irisin in the culture medium was determined using an ELISA kit. The results were presented as mean ± SEM. Statistical analysis of the data employed ANOVA and Fisher’s test for multiple comparisons, adopting significance level of 5% (p<0.05). Both time‐ and dose‐dependent effects on cell counts were observed. Culturing for 5 days led to a higher number of cells (111,367 ± 42,813 cells), compared to 3‐day culture (22,422 ± 9,141 cells). Furthermore, stimulation by ET‐1 at 10‐6 M resulted in higher numbers of cells for culture times of both 3 days (64,971 ± 41,841 cells) and 5 days (174,478 ± 49,152 cells), compared to the same culture times in the absence of ET‐1. Cell viability was higher for the 5‐day cultures of the control group (0.1984 ± 0.04) and the 10‐10 M ET‐1 group (0.2633 ± 0.0311), compared to the 3‐day cultures (control group: 0.1681 ± 0.003; 10‐10 M ET‐1 group: 0.1421 ± 0.0135). Biologically, in the absence of ET‐1, there was a time‐dependent reduction in irisin production (3‐day culture: 0.106 ± 0.0085 ng/mL; 5‐day culture: 0.097 ± 0.09 ng/mL). Therefore, the findings suggested that the multiplication activity of the C2C12 cells cultivated according to the proposed study model was modulated by time and by ET‐1, and that cell viability was modulated by the culture time. The time‐dependent decreased in irisin production suggested that there were negative effects on cell multiplication and viability, and that irisin could modulate the intracellular activity pathways for the mechanisms of cell differentiation.
Sex differences in cortical‐hypothalamic circuitry mediating cardiovascular disease risk
Authors: Schaeuble Derek , Wallace Tyler , Pace Sebastian A. , Myers Brent B.
Published: May 2022

Endocrine and autonomic stress responses adapt the body to real or perceived threats by engaging physiological pathways to maintain homeostasis. Unfortunately, the burden of prolonged stress contributes to maladaptive stress responding, increasing risk for pathologies including cardiometabolic disorders. However, the circuit mechanisms integrating endocrine and autonomic stress responses have yet to be defined. Our previous studies found that glutamate neurons of the infralimbic cortex (IL) have sex‐specific effects on endocrine and cardiovascular stress reactivity. While the IL is important for coordinating behavioral and physiological responses to stress, these cortical cells do not directly innervate the secretory or pre‐autonomic cells that initiate endocrine and autonomic stress responses. However, we previously found that the posterior hypothalamus (PH) receives dense stress‐activated inputs from the IL. Thus, the current study tests the hypothesis that IL glutamate release in the PH mediates sex‐specific effects on stress integration. To test this hypothesis, adult male and female Sprague‐Dawley rats received stereotaxic injections in the IL of a viral‐packaged construct expressing channelrhodopsin‐2 and bilateral fiber optics targeting the PH to stimulate IL synapses in the PH. After recovery and acclimatization to handling, rats were tested in the real‐time place preference paradigm to examine behavioral preference or aversion for circuit stimulation. These data indicate that IL‐PH signaling is preferred by males and that females have neither preference nor aversion for IL‐PH stimulation. Additionally, acute restraint was used to examine glucose and corticosterone responses to stress. Results found that IL synapses in the PH decrease stress‐induced glucose mobilization without affecting corticosterone in male rats. In contrast, IL‐PH stimulation in females increases corticosterone responses to restraint but does not affect glucose mobilization. Furthermore, non‐invasive assessment of hemodynamics indicate that IL‐PH stimulation increases heart rate in females but not males. Thus, the IL‐PH circuit increases place preference and reduces glucose responses to stress in males, while increasing corticosterone stress responses and heart rate in females. Taken together, these data suggest that the PH represents a key node for sex‐specific integration of descending cortical signals with endocrine and autonomic stress reactivity. Ultimately, sex‐specific delineation of stress‐regulatory neurocircuitry may provide insight into the differential impact of stress pathologies in men and women.
Antihypertensive Action of Exosomes Released from the Renal Medulla after Unclipping of the Renal Artery of 2 Kidney‐1 Clip Hypertension Mice
Authors: Hu Gaizun , Li Guangbi , Huang Dandan , Zou Yao , Yuan Xinxu , Li Pin‐lan
Published: May 2022

The renal medulla has been long known to exert antihypertensive action by releasing different bioactive substances. However, so far it remains unknown what is the identity of such renal medullary antihypertensive substances. In the present study, we hypothesized that exosomes, but not a single molecule released from the renal medulla are importantly implicated in the renal medullary antihypertensive action. To test this hypothesis, we first produced the 2 kidney‐one clip (2K1C) hypertension mouse model by clipping the right renal artery (127.9 ± 1.40 mmHg vs. 94.8 ± 1.75 mmHg of sham mice). After 1 week, we unclipped the right renal artery and monitored the mean arterial blood pressure (MAP) for 1 hour and at the same time collected blood samples for analysis of exosomes. It was found that unclipping the right renal artery of 2K1C mice significanty reduced MAP by 21.2 ± 2.65 mmHg but increased the plasma exosome concentrations by more than 5 folds (before unclipping 2.9 ± 0.7 vs. 3.3±0.5, N.S., after unclipping 4±0.5 vs. 17±5× 108particles/mL, p<0.05). Immunohistochemical (IHC) assessments showed that the clipped kidneys had dramatic increase in exosome markers (CD63 and Annexin II) in the renal medulla of 2K1C mice compared to contralateral kidneys or sham mouse kidneys. These functional and IHC changes in 2K1C mice were almost completely blocked by pretreatment of mice with an exosome biogenesis inhibitor, GW‐4869 (1 mg/kg/day, i.p., n=8). We also found that rapamycin, a compound that increases autophagy and lysosome trafficking (6 mg/kg/day, i.p., n=8) substantially attenuated exosome release from the renal medulla and associated antihypertensive action in 2K1C with unclipping the right renal artery, even though it had some effects to reduce MAP and increase exosome release before the unclipping. In another series of studies using 2K1C hypertensive mice, we extracted and purified exosomes from the blood of mice with unclipping of the right renal artery and then infused these exosomes intravenously to another group of 2K1C mice. It was found that plasma exosomes from 2K1C mice with unlipping of the right renal artery signicantly reduced MAP when they are infused into recipient 2K1C mice (at a concentration of 3 x 108 exosomes) compard to exosomes from sham mice. Urinary flow and sodium excretion of recipient mice also increased slightly by infusion of exosomes from 2K1C mice. These results suggest that exosomes are enriched in the renal medulla of the clipped kidney in 2K1C mice and they can be released into the blood if the renal artery clip is released. These exosomes have strong effects to lower arterial blood pressure.
Transferrin receptor‐mediated iron uptake is essential for colon tissue homeostasis
Authors: Kim Hyeoncheol , Xue Xiang
Published: May 2022

Background and aims

Transferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be downregulated via the iron regulatory protein and the iron‐responsive element machinery to reduce iron uptake and toxicity. However, the mechanism by which the expression of TFRC is maintained high in iron‐enriched cancer cells and its contribution to cancer development are enigmatic.

Methods

The regulation and function of TFRC‐mediated iron uptake in colon were tested in mouse models with colon‐specific TFRC disruption and colon‐derived cell lines. Transcriptome analysis of patient‐derived tumor colonoids was performed to identify molecular targets of iron.

Results

Our work shows TFRC is induced by adenomatous polyposis coli gene loss‐driven β‐catenin activation in colorectal cancer, whereas TFRC‐mediated intratumoral iron accumulation potentiates β‐catenin signaling via directly enhancing the activity of tankyrase. TFRC‐mediated iron import is at the center of this novel feed‐forward loop to facilitate colonic epithelial cell survival. Mechanistically, disruption of TFRC led to a reduction of colonic iron levels and iron‐dependent tankyrase activity, which caused stabilization of Axin2 and subsequent repression of the b‐catenin/c‐Myc/E2F1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation and TFRC disruption increased DNA replication stress, DNA damage response, apoptosis and reduced colon tumor growth. Strikingly, a combination of iron chelators and DNA damaging agents caused a synergistic effect in inducing DNA damage response and reducing colon tumor cell growth.

Conclusion

Together, the TFRC/iron/tankyrase/Axin2/b‐catenin/c‐Myc/E2F1/POLD1 axis is essential for colon homeostasis and may provide potential novel strategies for colorectal cancer therapy.
New Insights into the Structure‐Function Relationships of G Proteins Containing Disease‐Associated Mutations
Authors: Anazia Kara , Petrraco Enzo , Ferré Guillaume , Eddy Matthew T.
Published: May 2022

G proteins regulate intracellular signaling processes through their interactions with guanine nucleotides, GDP and GTP, and initiate numerous physiological responses. In normal conditions, G protein functions are regulated in a tightly controlled signaling network through complex formation with partner proteins, especially G protein‐coupled receptors (GPCRs). However, loss‐of‐function or gain‐of‐function mutations in the Gα subunit of G proteins disrupt this process, circumventing the usual control mechanisms and causing changes in G protein activity that result in serious illnesses. Our studies focused on the stimulatory class of trimeric G proteins, Gs, and mutations in Gαs associated with multiple cancers1. While the structure of one Gαs variant has been determined and additional Gαs variants have been characterized biochemically, there remains a need for an overall mechanistic framework for relating mutations in Gαs to changes in G protein signaling.

To fill in this gap in knowledge, we investigated the interactions of Gαs variants with nucleotides using saturation transfer difference nuclear magnetic resonance (STD‐NMR) spectroscopy in aqueous solutions. In STD‐NMR experiments, we observed interactions between nucleotides and the Gαs variant proteins at atomic resolution, which provided information on nucleotide binding modes. STD‐NMR data of one Gαs variant closely correlated with literature data of the structure and activity of the same variant protein2, establishing a foundation for extending the analysis of NMR data to seven additional disease‐associated Gαs variant proteins for which no structures are available. These NMR data were combined with correlative biophysical data that measured the overall fold of the Gαs‐nucleotide complexes and with functional data reporting on the relative affinities of the Gαs variants for GDP and GTP. Integrating these data together provided a conceptual framework for categorizing variations in Gαs amino acid sequences with changes in signaling activity. Surprisingly, differences in the binding modes of the Gαs variants did not always directly correlate with their different functions, suggesting that mutations in Gαs that impact nucleotide exchange are nuanced and complex. These results imply that strategies for the development of small molecule drugs may need to be specifically tailored for each disease associated Gαs variant.

References

O’Hayre, M. et. al. The emerging mutational landscape of G proteins and G protein‐coupled receptors in cancer. Nat. Rev. Cancer. 13, 412‐424 (2013).

Hu, Q. & Shokat, K. M. Disease‐Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP. Cell. 173, 1254–1264 (2018).
Sensitivity to Iron‐Mediated Pro‐Inflammatory Signaling in NZBWF1 Mice, a Model of Systemic Lupus Erythematosus
Authors: Dsouza Del L. , Boesen Erika I.
Published: May 2022

Introduction

The autoimmune disease systemic lupus erythematosus predominantly affects women and can lead to lupus nephritis (LN) as a common and severe complication in approximately 50% of patients. The F1 hybrid of New Zealand Black (NZB) X New Zealand White (NZW) mice (NZBWF1) is a well‐established mouse model to study LN. Our previous data indicated that the onset of albuminuria in female 34‐week NZBWF1 mice was associated with renal iron accumulation, and iron may contribute to renal injury. This study tests the hypothesis that 34‐week NZBWF1 lupus mice are sensitized to iron's injurious and pro‐inflammatory effects compared to 8‐week normal NZBWF1 mice. As a means to differentiate effects of age as opposed to development of lupus, parallel experiments were performed in 8 and 34‐week‐old NZW mice as healthy controls.

Methods and Results

Female 8‐week (pre‐autoimmune) and 34‐week NZBWF1 (autoimmune, pre‐albuminuric) mice together with age‐matched healthy control NZW mice underwent 24 h urine collection to confirm non‐albuminuric status. Mice were then were injected i.v. with a single dose of iron‐sucrose (2 mg elemental iron) or vehicle (saline) and sacrificed 24 h later. Plasma blood urea nitrogen (BUN) was measured to determine renal health, SRY‐Box Transcription Factor 9 (SOX‐9)‐ positive cells were quantified in kidney sections as an early tubular injury marker, and plasma monocyte chemoattractant protein‐1 (MCP‐1) was measured by ELISA as an inflammatory marker. Lupus (NZBWF1) and control mice (NZW) were compared within strains by age (8‐week vs. 34‐week) and treatment (Vehicle vs. Treatment). All statistical analyses were done by 2‐way ANOVA (n=7‐12/group), and post‐hoc testing was done using Bonferroni. Iron‐sucrose treatment significantly increased plasma MCP‐1 concentrations in the NZBWF1 and the control NZW mice (PTreatment < 0.01). There was no significant effect of age on this effect for NZW mice (PTreatment*Age = 0.2); however, in NZBWF1 mice, the iron‐sucrose induced increase in MCP‐1 was significantly greater in 34‐week compared to 8‐week mice (PTreatment*Age < 0.05; P<0.05 by posthoc test). BUN was significantly increased with age in both strains (PAge<0.05), but there was no significant effect of iron‐sucrose (PTreatment>0.05). The numbers of SOX‐9+ tubular cells increased in the 34‐week NZBWF1 mice compared to 8‐week mice (Page<0.05), consistent with early stages of renal injury, but iron sucrose treatment did not show any significant effect (PTreatment= 0.9).

Conclusion

Our data suggest that at the autoimmune but pre‐albuminuric stage, 34‐week NZBWF1 mice show increased pro‐inflammatory responsiveness to iron‐mediated stress. Further studies to determine whether the enhanced pro‐inflammatory effects of iron contribute to pathology in lupus nephritis are ongoing.
Epigenome‐wide association study of bronchopulmonary dysplasia (BPD) in preterm infants: Results from the Discovery‐BPD program
Authors: Cho Hye‐Youn , Wang Xuting , Campbell Michelle R. , Panduri Vijayalakshmi , Coviello Silvina , Caballero Mauricio T. , Sambandan Deepa , Kleeberger Steven R. , Polack Fernando P. , Ofman Gaston , Bell Douglas A.
Published: May 2022

Bronchopulmonary dysplasia (BPD) is a lung disease in premature infants characterized by impaired pulmonary development which persists into later life. While advances in neonatal care have improved survival rates of premature infants, cases of BPD have been increasing with limited therapeutic options for prevention and treatment. This study was designed to explore the relationship between gestational age (GA), birth weight and estimated blood cell‐type composition in premature infants and to elucidate early epigenetic biomarkers associated with BPD. Cord blood DNA from preterm neonates that went on to develop BPD (n = 14) or not (nonBPD, n = 93) was applied to Illumina 450K methylation arrays. Blood cell‐type compositions were estimated using DNA methylation profiles. Multivariable robust regression analysis elucidated CpGs associated with BPD risk. cDNA microarray analysis of cord blood RNA identified differentially expressed genes in neonates who later developed BPD. The need for oxygen supplementation was strongly associated (p < 1.0E‐09) with GA. The estimated nucleated red blood cell (NRBC) percent was negatively associated with birth weight and GA, positively associated with hypomethylation of the tobacco smoke exposure biomarker cg05575921, and high‐NRBC bloods displayed a hypomethylation profile. Epigenome‐wide association study (EWAS) identified 38 (Bonferroni) and 275 (false discovery rate 1%) differentially‐methylated CpGs associated with BPD. BPD‐associated CpGs in cord blood were enriched for lung maturation and hematopoiesis pathways. Stochastic epigenetic mutation burden was significantly elevated among those who developed BPD (adjusted p = 0.02). Methylation‐based GA prediction models suggested GA acceleration in BPD. Transcriptome changes in cord blood cells reflected cell cycle, development, and pulmonary disorder events in BPD. While results must be interpreted with caution because of the small size of this study, NRBC content strongly impacted DNA methylation profiles in preterm cord blood while EWAS revealed potential insights into biological pathways involved in BPD pathogenesis.
Cardioprotective effects of redox regulator MnTnBuOE‐2‐PyP 5+ in myocardial ischemia/reperfusion injury
Authors: Sharma Sudha , Patel Foram , Bhattarai Susmita , Ara Hosne , Subedi Utsab , Bhuiyan Md. Shenuarin , Batinic‐Haberle Ines , Panchatcharam Manikandan , Miriyala Sumitra
Published: May 2022

Background

Myocardial infarction is a leading cause of death worldwide and occurs due to decrease or complete blockage of coronary blood flow. Re‐establishment of blood flow after a brief period of ischemia is accompanied by exacerbation of the cardiomyocytes and its death, a phenomenon known as myocardial ischemia‐reperfusion (I/R) injury. Much evidence suggests that oxidative stress is involved in the pathogenesis of myocardial ischemia‐reperfusion injury and is potential target for therapeutic interventions. The aim of this study is to evaluate cardioprotective role of redox active Mn(III) N‐n‐butoxyethylpyridinium‐2‐yl)porphyrin, MnTnBuOE‐2‐PyP5+ (BMX‐001) in myocardial I/R injury. This drug is now in 4 Phase II clinical trials where the radioprotection of normal issue and anticancer efficacy have been evaluated.

Methods

Myocardial I/R injury in mice C57BL/6 was induced by ligating left anterior descending artery for 1 hour followed by 24 hours of reperfusion. At the end of reperfusion, hearts were sectioned (1mm) and stained with 1% 2, 3, 5‐Triphenyltetrazolium chloride (TTC) to determine infarct size. Cardiac function was evaluated by M‐mode echocardiography at the end of the reperfusion. For in vitro model, H9c2 cells were subjected to hypoxia for 3 hours followed by 24 hours of reperfusion. Cell apoptosis was measured by TUNEL assay. Mitochondrial superoxide and Cardiolipin peroxidation were measured by using MitoSOX and 10‐N‐nonyl‐Acridin Orange, respectively.

Results

In the current study, we demonstrated that treatment with MnTnBuOE‐2‐PyP5+ (2mg/kg) 4 hours before I/R injury in mice ameliorate cardiac injury as evident by limited infarct size and improved cardiac function (P<0.05). Consistently, MnTnBuOE‐2‐PyP5+ improved mitochondrial SOD2 expression (P<0.05) and reduced cardiomyocytes apoptosis in mice heart (P<0.05). In our in vitro model of hypoxia/reoxygenation injury in H9c2 cells, treatment with MnTnBuOE‐2‐PyP5+ (10µM) 24 hours before hypoxia/ reoxygenation injury decreased cardiolipin peroxidation, mitochondrial superoxide level and 4‐HNE adducted proteins (P<0.01). Additionally we explored role of MnTnBuOE‐2‐PyP5+ on cell apoptosis following ischemia/reperfusion injury in H9c2 cells. Redox regulator significantly reduced TUNEL positive cells following I/R injury indicating reduced cardiomyocytes apoptosis (p<0.05). In‐vitro, redox‐active Mn porphyrin improved cell viability and lactate dehydrogenase release following hypoxia/reoxygenation in H9c2 cells (p<0.05).

Conclusion

This result provided evidence that MnTnBuOE‐2‐PyP5+ elicited cytoprotective effects through reduction of mitochondrial oxidative stress and cardiomyocyte apoptosis suggesting prospective therapeutics in myocardial I/R Injury.
Cocoa And Cocoa Polyphenol Fractions Fails To Improve Colitis In Dextran Sulfate Sodium‐Treated Mice
Authors: Weikart Daphne K. , Lambert Joshua
Published: May 2022

Cocoa and cocoa polyphenols have been shown to have anti‐inflammatory effects in the context of obesity and cardiometabolic disease in both animal models and human, however the anti‐inflammatory effects have not been well‐studied in models of inflammatory bowel disease (IBD). The aim of this study was to compare the anti‐inflammatory effects of cocoa and cocoa polyphenol fractions in the dextran sulfate sodium (DSS)‐induced mouse model of colitis. We hypothesized that cocoa and cocoa polyphenols would exert anti‐inflammatory effects in this model. Male mice were treated with defatted cocoa powder (8%) or the equivalent dose of solvent extractable polyphenols or non‐extractable materials. After 2 weeks of dietary supplementation, mice were given 2.5% DSS as the sole source of drinking water for 1 week prior to euthanasia. Treatment with cocoa or cocoa fractions had no significant effect on spleen weight or colon length, both gross markers of inflammation. DSS treatment increased mRNA expression of interleukin (IL)‐ 6, ‐10, ‐1β, and tumor necrosis factor (TNF)‐ α; no significant difference in the expression of these markers was observed between the cocoa supplementation groups and the DSS controls. In summary, cocoa and cocoa polyphenols do not exert an anti‐inflammatory effect in the DSS‐induced mouse model colitis and may not be effective against IBD.
Effects of Diallyl Trisulfide on TNF‐α‐stimulated Genetically different Triple‐Negative Breast Cancer Cells
Authors: kanga Konan W. , Mendonca Patricia , Soliman Karam F. A. , Ferguson Dominique , Darling‐Reed Selina
Published: May 2022

Many studies have demonstrated the potential anticancer properties of garlic and their respective constituents through in vitro and in vivo studies. Diallyl trisulfide (DATS), an organosulfur compound of garlic, has been shown to prevent and inhibit breast carcinogenesis through various signaling pathways. The triple‐negative breast cancer (TNBC) subtype, which accounts for approximately 15% of all breast cancers, usually behaves more aggressively than other types of breast cancer and is more challenging to treat. Cancer progression is associated with the overexpression of the monocyte chemotactic protein‐1 (MCP‐1. MCP‐1, also known as C‐C motif chemokine ligand‐2 (CCL2), plays a significant role in breast cancer cell signaling, leading to increased cell proliferation, immune suppression, epithelial‐to‐mesenchymal transition (EMT), and angiogenesis. This study explored DATS effects on the genetically different triple‐negative breast cancer cells, MDA‐MB‐231 and MDA‐MB‐468. DATS effects on TNF‐α induced TNBC cells were examined via trypan blue exclusion test, wound‐healing assay, human cytokine arrays, ELISA, and RT‐PCR. The results showed that DATS induced cytotoxicity in a dose and time‐response way in MDA‐MB‐231 and MDA‐MB‐468 cell lines. Wound healing assays demonstrated that DATS significantly inhibited cell migration after a 12 h exposure in both cell lines. Protein expression of CCL2/MCP‐1, IL‐6, PDGF‐BB, NT‐3, and GM‐CSF was increased in the TNF‐α‐treated cells. However, the cotreatment with DATS and TNF‐α showed that the compound significantly decreased the expression of CCL2/MCP‐1 in MDA‐MB‐231 but not in MDA‐MB‐468 cells; data confirmed with ELISA. Moreover, DATS significantly down‐regulated mRNA expression of IKBKE and MAPK8 in both cell lines, indicating a possible effect in genes involved in the NF‐κB and MAPK signaling. The data show that genetically different cells may respond in a different way to DATS treatment. In conclusion, DATS may be a potential candidate for breast cancer therapy to slow TNBC progression.
Identification of Disease‐relevant, Sex‐based Proteomic Differences in iPSC‐derived Vascular Smooth Muscle
Authors: Ariyasinghe Nethika R. , Escopete Sean , De Souza Santos Roberta , Gross Andrew , Sareen Dhruv , Parker Sarah J.
Published: May 2022

The prevalence of cardiovascular disease varies with sex, and the impact of intrinsic sex‐based differences on vasculature is not well understood. As animal models have limited utility in the exploration of sex‐based differences in humans, we used iPSC‐derived vascular smooth muscle cells from healthy patients of both sexes to identify sex‐based proteomic differences and their possible effects on cardiovascular pathophysiology. Our analysis confirmed that differentiated cells have a proteomic profile more similar to healthy primary aortic smooth muscle than iPSCs. We also identified sex‐based differences in iPSC‐derived vascular smooth muscle in pathways related to integrins, extracellular matrix proteins, focal adhesions, and metabolism of amino acids. Exploration of these pathways can lead to the discovery of sex‐specific pharmacological targets for cardiovascular disease.
DCT‐specific COP9 Signalosome Deletion Activates the WNK4‐NCC Pathway and Mimics Familial Hyperkalemic Hypertension
Authors: Cornelius Ryan J. , Su Xiao‐Tong , Yang Chao‐Ling , Ellison David H.
Published: May 2022

With‐no‐lysine kinase 4 (WNK4) activates the NaCl cotransporter (NCC) which is the main pathway for sodium reabsorption in the distal convoluted tubule (DCT) of the kidney. The cullin‐RING ubiquitin ligase cullin 3 (CUL3) regulates WNK4 abundance via interaction through the substrate adaptor KLHL3. The monogenic disease familial hyperkalemic hypertension (FHHt) is caused by mutations in this pathway. Mutations in KLHL3 and WNK4 mainly disrupt formation of this complex, whereas, the CUL3 mutations impair binding to the COP9 signalosome (CSN), a deneddylase and upstream regulator of cullin‐RING ligases. Since FHHt is mostly a disease that affects the DCT, we proposed that DCT‐specific knockdown of the main CSN subunit, JAB1, would phenocopy FHHt. We deleted Jab1 in the DCT using the tamoxifen inducible NCC‐Cre‐ERT2 mouse model (DCT‐Jab1‐/‐). After five days of tamoxifen administration, proximal tubule, thick ascending limb, DCT, and connecting tubule/collecting duct segments were individually dissected and isolated to analyze protein abundance of each specific segment via Western blot. JAB1 and CUL3 protein abundance were substantially reduced in the DCT of DCT‐Jab1‐/‐ mice, whereas, WNK4 and phosphorylated NCC (pNCC) abundance were markedly elevated. Western blot analysis of cortical kidney tissue showed lower KLHL3 abundance and significantly higher abundance of phosphorylated SPAK (pSPAK), the intermediary kinase between WNK4 and NCC. Immunofluorescent staining of WNK4 and pSPAK showed increased expression and puncta formation in DCT1 and DCT2 tubules of DCT‐Jab1‐/‐ mice. This was not observed in other tubule segments that express WNK4 or pSPAK, such as the thick ascending limb and connecting tubule/collecting duct. Although there was obvious activation of the WNK4‐SPAK‐NCC pathway DCT‐Jab1‐/‐ mice, plasma analysis showed no significant differences in potassium, chloride or TCO2. However, challenging the DCT‐Jab1‐/‐ mice with a high potassium diet for seven days exaggerated the differences in pSPAK and pNCC protein abundance and led to a significant increase in plasma potassium. The results indicate that DCT‐specific disruption of JAB1 recapitulates many aspects of FHHt, including decreased KLHL3 and CUL3 abundance which leads to activation of the WNK4‐NCC pathway and a tendency toward hyperkalemia. These results implicate defective CSN binding as a key component of CUL3‐mediated familial hyperkalemic hypertension.
Effects of neutral sphingomyelinase activation associated factor (Nsmaf) gene knock‐down on myogenesis in murine C2C12 myoblast
Authors: Jung Jay , Sumi Koichiro , Kouzaki Karina , Tamura Yuki , Kotani Takaya , Nakazato Koichi
Published: May 2022

Neutral sphingomyelinase activation associated factor (Nsmaf) modulate ceramide production by activating neutral sphingomyelinase enzymes. Nsmaf also regulates tumor necrosis factor‐α (TNF‐α)‐induced signaling pathway. Since both ceramide metabolisms and TNF‐α signaling are key events during inflammation, Nsmaf might play a nodal role in inflammation‐induced events, such as muscle wasting. In the present study, we examined whether Nsmaf gene knock‐down effects on myogenesis in murine C2C12 myoblasts. C2C12 myoblasts were used in this study. In the growth phase, cells were maintained in DMEM and 10% FBS (growth medium: GM). For cell differentiation, the culture medium was switched to DMEM containing 1% HS (differentiation medium: DM). Small interfering RNA (siRNA) was used to induce Nsmaf gene Knock‐down (NsmafKD, n = 6). siRNA with a scrambled sequence was used as negative controls (Scr, n = 6). Gene knockdown was performed both in myoblasts and myotubes. After two‐day siRNA treatment, Western blotting was performed to examine protein expression levels. Laser scanning microscopy was used to investigate cell morphology. Even in the GM condition, differentiation markers, such as myogenin and myosin heavy chain (slow) were upregulated in the NsmafKD cells. In transfected myotubes, myotube diameter of the NsmafKD was about 50% smaller than that of the Scr. Both in myoblasts and myotubes, contents of a cell fusion mediator, myomixer, in the NsmafKD was significantly lower than that in the Scr. These data indicate that Nsmaf is a novel regulator of myogenesis.
Interferon‐alpha or ‐beta Facilitates SARS‐CoV‐2 Pulmonary Vascular Infection by Inducing ACE2
Authors: Yuan Ke , Klouda Timothy , Yuan Hao , Kim Hyunbum , Kim Jiwon , Olejnik Judith , Hume Adam J. , Melero‐Martin Juan , Mühlberger Elke , Jia Hongpeng , Padera Robert F. , Raby Benjamin A.
Published: May 2022

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is characterized by a hyperinflammatory state typified by elevated circulating pro‐inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID‐19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS‐CoV‐2 binding receptor ACE2. Herein we describe SARS‐CoV‐2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID‐19, demonstrating both heterogeneous ACE2 expression and endothelial damage (Figure). In primary endothelial cell cultures, we show that SARS‐CoV‐2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon‐alpha (IFNα) or ‐beta(β) ‐ two of the main anti‐viral cytokines induced in severe SARS‐CoV‐2 infection ‐ but not significantly by other cytokines (including interleukin 6 and interferon g /λ). Our findings suggest that the stereotypical anti‐viral interferon response may paradoxically facilitate the propagation of COVID‐19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID‐19.
Unraveling Molecular and Metabolic Gut‐Brain Signalling Disruptions and Potential Therapy in New Bardet‐Biedl Syndrome Mouse
Authors: Singh Arashdeep , Haq Naila , Luckey Shelby , Mansouri Samira , Gogoi Himanshu , Rourk Katelyn , Jin Lei , Christou‐Savina Sofia , de Lartigue Guillaume
Published: May 2022

Obesity is a cardinal feature of Bardet‐Biedl syndrome (BBS), a syndromic ciliopathy caused by mutations in genes for BBS 1‐21. BBS proteins control the function of primary cilia that act as a sensory antenna on virtually all mammalian cells. The mechanisms of BBS‐induced obesity remain poorly understood, resulting in a lack of therapeutic options for these patients. The overarching goal of the study was to define underlying mechanisms involved in the pathogenesis of BBS‐induced obesity and use this knowledge to identify novel therapeutic targets. Given the importance of cilia in interoceptive and homeostatic sensing, we hypothesized that disruption of cilia function by BBS mutation would impair negative feedback resulting in a proinflammatory microenvironment in adipose tissue and disruption of neuroendocrine control of feeding in hypothalamus.

In this study, we first characterized the metabolic phenotype of a novel BBS5 knockout mouse (BBS5‐/‐; n=6‐12/strain/gender). Next, we performed immunophenotyping of adipocytes and unbiased RNA sequencing of the hypothalamus of BBS5‐/‐ and WT mice. To validate the predictions from transcriptomic data, we performed pharmacological‐based feeding experiments and tested a putative therapeutic for BBS‐induced obesity.

We found that BBS5‐/‐ mice exhibits all the classic symptoms observed in human patients with BBS, including hyperphagia, cognitive impairments, glucose, and insulin intolerance, and 3 to 4‐fold increased fat mass on chow diet compared to wildtype mice (p<0.001). BBS5‐/‐ mice had increased proinflammatory M1 macrophages and decreased functional regulatory T‐cells in adipocytes suggesting that extrinsic factors promote fat mass gain. Pathway analysis of the transcriptomics data revealed altered endocrine receptor signaling in the hypothalamus of BBS5‐/‐ mice. Feeding experiments confirmed the development of both leptin and cholecystokinin resistance independent of body weight (p>0.05). The receptor for glucagon‐like‐peptide‐1 (GLP1) was found to be upregulated in the hypothalamus of BBS5‐/‐ mice (p<0.001). Chronic daily pharmacological administration of GLP1 receptor agonist (Semaglutide) for two weeks significantly reduced food intake, body weight, elevated glucose levels, and fat mass (p<0.001).

In conclusion, BBS5‐/‐ mice are a valuable new translational tool to elucidate interoceptive and homeostatic gut‐brain axis alterations in BBS‐induced obesity. We identify adipose inflammation, and impaired hypothalamic leptin, insulin, and cholecystokinin signalling as underlying mechanisms for the metabolic pathogenesis of ciliopathy. Our results support using GLP1 receptor agonist as a novel therapeutic strategy for treating obesity in patients with BBS.
Deacetylated Sialic Acids Increases Selectin‐binding in Lung Cancer Cells
Authors: Schulte Megan , Willand‐Charnley Rachel
Published: May 2022

Cancers utilize glycans to evade the immune system via the Sialic acid‐ Selectin pathway. Sialic acid specifically uses E‐ selectins, P‐ selectins, and L‐ selectins which each have different properties and preferred ligands. Here, we focused on modulating 9‐O and 7, 9‐O‐acetylation of Neu5Ac, via CRISPR‐ Cas9 gene editing, a functional group that is absent from Sias on many types of cancer cells. The two genes that are responsible for regulating the level of acetylation on Neu5Ac, are Sialic acid acetylesterase (SIAE) and Sialic acid acetyltransferase (CASD1). These genes modulated Selectin binding in lung cancer cells. In the absence of SIAE, Neu5Ac is acetylated, and binding of selectins is slightly increased whereas binding was greatly increased when the ability to acetylate was removed via CASD1 knock out. In addition to modulation Selectin binding, the absence of CASD1 knock out showed a greater expression of both PSGL‐1 (P‐selectin glycoprotein ligand) and Sialyl‐Lewisx. These glycans are essential ligands for P‐ and L‐ selectins, respectively. We are currently working on migration data to see how the modulation of Sialic acid contributes to the metastatic property of lung cancer. Uncovering how functional group alterations on Neu5Ac contribute mechanistically to both Selectin receptor binding, the Sialic Acid‐Selectin immune evasion pathway, Selectin‐receptor mediated migration, and the production of cancer‐associated selectin ligands—offers a promising avenue for targeted cancer immune therapies in the future.
The miR‐200 family targets Tbx5, Gata4, and Pitx2during cardiac development and regeneration
Authors: Leonard Riley J. , Sweat Mason , Eliason Steven , Amendt Brad A.
Published: May 2022

Cardiac development requires a highly complex gene regulatory network (GRN) of transcription factors. These GRN factors are known to be modulated by microRNAs (miRs), small non‐coding RNAs that target mRNAs to alter expression levels. MiR‐200 family members, miR‐200aand ‐200c­, are highly expressed in the developing heart. These miRs are known to target cardiac transcriptions factors Tbx5, Gata4, and Pitx2. However, no in vivoinvestigation of the relationship between the miR‐200family members and these transcription factors has been done to date. We hypothesize miR‐200 family members modulate Tbx5, Gata4, and Pitx2expression to allow for proper cardiac development to occur. To study the role of miR‐200 family members, we developed the Plasmid‐based miR Inhibitor System (PMIS). PMIS works by repressing miRs through seed sequence complementation, allowing for the study of individual miR family members within a cluster. Histological analysis of PMIS‐miR‐200a, PMIS‐miR‐200c, and PMIS‐miR‐200ac hearts revealed distant abnormal phenotypes, with PMIS‐miR‐200achearts presenting with a ventricle‐septal defect. Additionally, we observe embryonic stage and chamber specific changes in Tbx5, Gata4, and Pitx2 expression when miR‐200aand ‐200c are inhibited. These aberrant expression changes result in altered expression of Tbx5, Gata4, and Pitx2 downstream transcriptional targets Nppa, Gja1, and Ace2. Tbx5, Gata4, and Pitx2 drive transcription of miR‐200a and ‐200c, resulting in a negative feedback loop on to these transcription factors. Post‐natal day 1 PMIS‐miR‐200a and PMIS‐miR‐200cpups have functional and structural phenotypes which are fully recovered by postnatal day 7. We discovered a role for miR‐200aand ‐200c in cardiac development through targeting Tbx5, Gata4, and Pitx2, with potential for using PMIS in therapeutic applications.
Transformations of phosphonates by non‐heme iron‐dependent oxygenases
Authors: Zechel David
Published: May 2022

Organophosphonates (Pns) comprise a prominent class of natural products that is defined by a highly stable C‐P bond. The biochemistry of Pns has evolved into two worlds, with one dedicated to synthesizing the C−P bond and incorporating this motif into complex bioactive molecules, while the other seeks to cleave the C−P bond to release inorganic phosphate, a life limiting nutrient. Non‐heme iron‐dependent oxygenases play important roles in both biosynthetic and catabolic Pn pathways. This presentation will cover recent advances in elucidating new functions of such enzymes in the oxidation of C‐P and C‐H bonds in Pns.