bluuebunny/crossref_metadata_2025 huggingface dataset:
Tobramycin Adaptation Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa Quorum Sensing‐Null Mutants
Authors: Townsend Kade A. , Abisado Rhea , McKee Brielle , Chandler Josephine R.
Published: May 2022

The bacterium Pseudomonas aeruginosa causes serious disease in immunocompromised patients and is a model for studying quorum sensing, a cell‐cell signaling system that becomes activated at a certain population or “quorum.” The P. aeruginosa master quorum sensing regulator is LasR, which drives transcription of dozens of genes including those coding for virulence and antibiotic resistance. Paradoxically, lasR‐null mutants are common in infections of tobramycin‐treated patients, presenting a challenge to ongoing efforts to develop anti‐LasR therapeutics. To understand how these mutants might arise, we performed a laboratory evolution experiment. We grew P. aeruginosa populations with sublethal tobramycin. Every day, we transferred the population to fresh medium and every three days we increased the tobramycin concentration. Variants from these populations became highly tobramycin resistant. Surprisingly, inactivating LasR in some of the variants further increased resistance, which was the opposite effect as that observed in the ancestral strain where LasR inactivation decreased tobramycin resistance. This phenomenon is known as sign epistasis. The evolved variants with the altered LasR phenotype all encoded a point mutation in the translation elongation factor fusA1. fusA1frequently incurs mutations as a mechanism of adaptation to tobramycin in antibiotic‐treated patient infections. We showed that the fusA1 mutation reversed the effect of LasR on antibiotic resistance, and thus is responsible for sign epistatic effects on LasR. Our results suggest that antibiotic selection might drive the accumulation of mutations that reverse the effect of LasR mutants on tobramycin resistance. These results could possibly explain how lasR mutants emerge in infected patients and have implications for developing novel therapeutics to treat antibiotic‐resistant infections.
The Loss of Endothelial Glycocalyx in Diabetic Retinopathy
Authors: Kaur Gaganpreet , Song Yuefan , Xia Ke , Cruz‐Topete Diana , McCarthy Kevin , Zhang Fuming , Linhardt Robert , Harris Norman
Published: May 2022

Introduction

The vascular endothelium is lined with the endothelial glycocalyx, composed of core proteins and glycosaminoglycans (GAGs). The loss of glycocalyx can cause endothelial injury and could contribute to the progression of retinopathy. The glycocalyx thickness has been shown to decrease in the diabetic retina; however, changes in individual components of the retinal glycocalyx have yet to be determined. Therefore, the present study examined hyperglycemia‐induced changes in retinal glycocalyx components.

Methods

Type‐1 diabetic rats and primary rat retinal microvascular endothelial cells (RRMECs) exposed to high glucose were used to mimic diabetes. Western blots and qRT‐PCR were used to analyze the expression of core proteins. Further, GAG composition was studied using LC‐MS.

Results

Both retinal endothelial cells and rat retina exhibited a significant decrease in mRNA transcripts and protein levels of syndecan‐3 under hyperglycemic condition, whereas syndecan‐1 expression was significantly increased. Further, we observed a substantial loss of glypican‐1 in RRMECs with high glucose, but a significant increase in the retina of diabetic rats. RRMECs in high glucose exhibited a significant decrease in chondroitin sulfate (CS) and hyaluronic acid (HA), but no change in heparan sulfate (HS) levels. In addition, 4‐O‐sulfated CS residues and 2‐O‐N‐sulfated HS residues were reduced, whereas N‐sulfated and 6‐O‐N‐sulfated HS residues were increased in retinal endothelial cells treated with high glucose. Lastly, we observed a significant increase in media levels of syndecan‐1, HA, HS, and CS, indicating increased shedding of glycocalyx components under hyperglycemia.

Conclusions

Our findings suggest that the loss of endothelial glycocalyx in the diabetic retina could be a result of a loss of the proteoglycan syndecan‐3 (and possibly glypican‐1) as well as the GAGs CS and HA.
Age‐dependent differences in the ventilator‐induced diaphragm dysfunction (VIDD): A multi‐omics study
Authors: Wen Ya , Lyu Qiong , Zhang Xiang , Larsson Lars
Published: May 2022

Introduction & Objective

Mechanical ventilation is a life‐saving intervention in patients with respiratory failure. Controlled mechanical ventilation (CMV) is the mode of mechanical ventilation used in patients with an insufficient central respiratory drive and frequently used in COVID‐19 patients ventilated in the prone position. However, CMV is also associated with negative consequences for the major respiratory muscle the diaphragm, i.e., the ventilator induced diaphragm dysfunction (VIDD). VIDD is the major factor underlying the delayed weaning from the ventilator especially in old intensive care unit (ICU) patients. The age‐related difference in mortality rate has become a major concern during the COVID‐19 pandemic. The current experimental study was undertaken to improve our understanding the mechanisms underlying age‐related differences in the response to CMV using a unique experimental ICU model allowing long‐term studies in a rat exposed CMV.

Materials and Methods

Young (7‐8 months) and old (28‐32 months) female F344 BN hybrid rats were exposed to the ICU condition for 5 days (anaesthetized, mechanically ventilated, and pharmacologically paralyzed by post‐synaptic blockade of the neuromuscular transmission). Transcriptomics (RNA‐Seq) and proteomics (Olink) analyses of the diaphragm and proteomics analysis of plasma were conducted to investigate the molecular differences between young and old rats exposed to the ICU condition.

Results

According to multi‐omics analyses, significant differences were observed in the diaphragm between young and old rats in response to ICU condition. In the diaphragm of young rats, CMV resulted in decreases in the biological processes related to energy metabolism and production as indicated by the downregulation of pathway like “glycolysis/gluconeogenesis”, “fatty acid metabolism”, “TCA cycle”, and “oxidative phosphorylation”, etc. On the other hand, in the diaphragm of old rats, dramatic immune and inflammatory responses were observed after CMV, as reflected in the upregulation of “Cytokine‐cytokine receptor interaction”, “TNF signaling pathway”, “IL‐17 signaling pathway”, etc. Transcriptomic results were consistent with plasma proteomics analyses, i.e., CMV induced a higher level of inflammatory factors and cytokines/chemokines in old rats.

Conclusions

After 5‐days exposure to CMV, our multi‐omics study demonstrated age‐related inflammatory response to long‐term mechanical ventilation.

Significance/Implication

Thus, CMV should be cautiously used and restricted to as short duration as possible, especially in the old patients. In the current COVID‐19 pandemic, the dramatically increased mortality in old ICU patients with COVID‐19‐associated hyperinflammation and cytokine storm need not only reflect the viral infection but may also be associated with the VIDD and hyperinflammatory responses induced by CMV per se.
The Regulatory Role of miR‐21 in Coronary Microcirculation
Authors: Juguilon Cody , Wang Yang , Wang Zhiyuan , Enrick Molly , Gadd James , Clark Alyssa , Chilian William , Yin Liya
Published: May 2022

Introduction

Coronary microvascular dysfunction is characterized by impaired endothelial‐dependent vasodilation. These impairments are seen in diabetic cardiomyopathy (DCM), coronary artery disease (CAD) and ischemia with non‐obstructive coronary artery (INOCA), Takotsubo cardiomyopathy, myocardial infarction with non‐obstructive coronary artery disease (MINOCA), and heart failure with preserved ejection fraction (HFpEF), but detailed mechanisms have yet to be elucidated.

Methods

microRNA‐21 (miR‐21) global and conditional knockout mice were used to study how miR‐21 regulates coronary microcirculation in pathological conditions like DCM. Both genetic (db/db) and diet‐induced diabetic models were used. Coronary arteries were isolated, and endothelial‐dependent vasodilation was assessed using myography (DMT). In vivo myocardial blood flow (MBF) under stress was measured by contrast echocardiography or doppler after the treatment with different dosages of norepinephrine. Quantitative polymerase chain reaction (qPCR) was performed for gene expression analysis. Trichrome staining and histology were performed for structural changes of the hearts.

Results

Our preliminary data show that miR‐21 is upregulated in DCM and the deficiency of miR‐21 restores the endothelial‐dependent vasodilation in isolated diabetic coronary arterioles and coronary blood flow under stress in DCM through the mechanism that miR‐21 prevents the mediator of coronary vasodilation switching from NO to H2O2 in diabetes.

Conclusions

miR‐21 regulates microvascular dysfunction in DCM. Further genetic profiling will elucidate the pathways and mechanisms converging with miR‐21 to regulate microvascular function.
Kininase I Inhibition Prevents Angiotensin II‐Induced Hypertension and Target Organ Damage
Authors: White Acacia , Parekh Rohan U. , Sriramula Srinivas
Published: May 2022

Activation of the kinin B1 receptor (B1R), a major component of the kallikrein kinin system (KKS), is associated with the pathogenesis of hypertension. Kininase I (or carboxypeptidase N/M; CPN/CPM), an enzyme primarily responsible for the synthesis of endogenous B1R agonist, has been implicated in the progression of cardiovascular diseases. However, the exact role of kininase I in hypertension has not been investigated. Our preliminary data shows that CPN activity is significantly increased in the brain of angiotensin (Ang) II‐induced hypertensive mice compared to vehicle treated mice (Ang II: 7.1 +0.4 AU/min vs. vehicle: 2.8 +0.3 AU/min, n=5, p<0.05). In this study, our objective was to clarify the role of kininase I in the pathogenesis of hypertension, particularly the effects of this enzyme on blood pressure, cardiac and renal hypertrophy. We hypothesized that inhibition of kininase I will attenuate the development of Ang II‐induced hypertension and target organ damage. To test this hypothesis, 12‐week old male and female C57BL/6NJ wild‐type (WT) and CPN gene deleted (CPNKO) mice, were administered Ang II (600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. Blood pressure was measured using radio telemetry probes and hypertrophy was assessed in the heart and kidney after 14 days of Ang II‐infusion, using weight/tibial length. Baseline blood pressure was similar between WT and CPNKO mice (102 +2 mmHg vs. 104 +2 mmHg). Ang II‐infusion significantly increased the blood pressure in WT mice compared to saline infused mice (147 +5 mmHg, n=5, p<0.05). This Ang II‐increased blood pressure response was attenuated in CPNKO mice (125 +4 mmHg, n=5, p<0.01). Both male and female CPNKO mice did not show any significant differences in blood pressure following Ang II‐infusion. In addition, Ang II‐infusion also induced significant cardiac (heart weight/tibia length: 8.5 +0.9 vs. 6.8 +0.6 mg/mm) and renal (kidney weight/tibia length: 11.8 +1.5 vs. 8.7 +0.9 mg/mm) hypertrophy in WT mice compared to vehicle treated mice. However, CPNKO mice exhibited an attenuation in cardiac and renal hypertrophy following Ang II administration. Furthermore, Ang II‐infusion increased collagen deposition in the heart, measured by picrosirius red staining, which was attenuated in CPNKO mice (percent area 9.8 +1.4 vs. 7.2 +1.4). Ang II‐infusion also significantly increased B1R gene and protein expression in the hypothalamus, heart, kidney, and aorta of WT mice, but this expression was abrogated in CPNKO mice. In conclusion, our data suggest that CPN gene deletion protects from Ang II‐induced hypertension and target organ damage, and indirectly prevents the B1R activation. This is the first evidence to show that inhibition of kininase I prevents the development of Ang II‐induced hypertension. Targeting kininase I might be a novel therapeutic approach for the treatment of hypertension.
Pyllelic, a Software Suite for Examining Allelic DNA CpG Methylation Patterns in Genomic Datasets
Authors: Bonham Andrew J. , Poch Dylan , Rowland Teisha J.
Published: May 2022

While genome‐wide demethylation of DNA CpG sites is a well‐established early hallmark of cancer, only recently has the presence of allelic methylation patterns associating with pathological gene expression been reported. Such allelic methylation patterns have often been obscured due to statistical averaging procedures applied to bisulfite conversion sequencing (Bis‐Seq) CpG methylation genomic datasets. Here, we present a Python software suite, Pyllelic, that dissects Bis‐Seq genomic datasets at the individual read level, allowing nuanced analyses of methylation patterns of genes, including allelic patterns associated with gene expression. We used this approach to examine the role of allelic methylation in the telomerase reverse transcriptase (TERT) gene, which is pathologically expressed in the majority of human cancers (approximately 80‐90%) and has known allelic expression patterns. Surprisingly, active TERTgene expression in human cancers has been characterized as having a hypermethylated CpG island in the promoter, which is the opposite of general promoter methylation associations with gene expression, including reports of hypomethylation of promoters correlating with overexpression of oncogenes. However, when we used Pyllelic to analyze >600 cancer cell lines from 23 different tissue types, we found that in the proximal region of the TERTpromoter, allele‐specific hypomethylation correlated with TERTexpression. This analysis was performed in cancer cell lines with known activating, monoallelic promoter mutations as well as in wild‐type TERTcancer cell lines. The Pyllelic suite allows both aggregate and individual read‐level access to the underlying methylated reads of genomic datasets, with a data science‐focused implementation to facilitate the path from hypothesis to publishable figures and statistical analyses. Pyllelic thus should serve as an important tool to help identify allelic DNA methylation patterns and associations for a wide variety of genomic regions, with particular relevance for pathological activation in cancers, as well as other tissue or disease methylation analysis.
Cyclosporine A but not tacrolimus induces pro‐apoptotic endoplasmic reticulum stress in renal tubular cells
Authors: Yilmaz Duygu E. , Kirschner Karin , Demirci Hasan , Himmerkus Nina , Bachmann Sebastian , Mutig Kerim
Published: May 2022

Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first‐line immunosuppressive strategies in organ‐transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro‐apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro‐apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4‐PBA, alleviated the CsA‐induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9‐mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro‐apoptotic UPR in response to CsA.

In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro‐apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity.
Targeting Hypoxia‐Adenosinergic Signaling to Enable Effective Anti‐Tumor Responses by Reprogramming the Immunosuppressive Tumor Microenvironment
Authors: Beattie Kai , Romero Navarrete Nuria Roxana , Dongre Anushka , Sitkovsky Michail , Hatfield Stephen
Published: May 2022

The hypoxia‐HIF‐1ɑ‐mediated generation of extracellular adenosine (eADO) and associated A2A adenosine receptor (A2AR) signaling represent a critical mechanism for resistance to anti‐tumor immune attack in an immunosuppressive tumor microenvironment (TME). This is supported by earlier clinical and preclinical findings that tumors exhibiting refractory responses to chemotherapeutic and immunotherapeutic interventions are often characterized by low tissue oxygen tension and elevation of intracellular cAMP triggered by the binding of adenosine to the A2AR. The ensuing signaling cascade results in the downregulation of T cell activation and increased production of anti‐inflammatory factors. Several classes of drugs have been proven in preclinical studies and clinical trials to weaken this powerful biochemical mechanism of immunosuppression and enable tumor rejection. Therapeutic approaches targeting the upstream and downstream stages of hypoxia‐adenosinergic negative immune regulation (i.e. A2AR antagonists, oxygenation, inhibition of adenosine generation or adenosine degradation) represent promising co‐adjuvants capable of inducing a more immunopermissive TME. The removal of this biochemical barrier may be the key to unleashing the full therapeutic potential of immunotherapies. For instance, the epithelial‐mesenchymal transition (EMT) program, which confers the ability of cancer cells to engage in metastatic dissemination, is associated with the acquisition of resistance to immune checkpoint blockade (ICB). Recent studies have demonstrated that abrogation of the adenosine‐generating ectoenzyme CD73 sensitizes mice bearing quasi‐mesenchymal (qM) tumors to anti‐CTLA4 ICB through an increase in CD8+ T cell infiltration and cytotoxicity. Furthermore, the present study demonstrates the differential expression of adenosinergic enzymes and EMT transcription factors (EMT‐TFs) in murine breast epithelial (E) and qM carcinoma cell lines cultured in normoxic (21% oxygen) and hypoxic (1% oxygen) conditions in vitro. As hypothesized, the stabilization of HIF‐1ɑ in hypoxic conditions resulted in the upregulation of some adenosine‐generating enzymes and changes in adenosine metabolism. These results elucidate an additional mechanism through which hypoxia can contribute to the assembly of an immunosuppressive TME, while highlighting the categorical differences between the E vs qM states of carcinoma cells.
Aging Selectively Reduces Mitochondrial Volume Density and Respiratory Capacity in Type IIx/IIb Diaphragm Muscle Fibers
Authors: Davis Leah A. , Brown Alyssa D. , Fogarty Matthew J. , Mantilla Carlso B. , Sieck Gary C.
Published: May 2022

Diaphragm muscle (DIAm) sarcopenia involves selective atrophy of type IIx/IIb DIAm fibers. In addition, there is an age‐related decrease in the maximum velocity of the succinate dehydrogenase reaction (SDHmax) in type IIx/IIb fibers. SDH is a key enzyme of the tricarboxylic acid (TCA) cycle as well as complex II of the electron transport chain (ETC). These results suggest that sarcopenia may reduce mitochondrial volume density in type IIx/IIb DIAm fibers and thereby reduce SDHmax. Alternatively, sarcopenia may reduce the intrinsic respiratory capacity of mitochondria in type IIx/IIb DIAm fibers. We hypothesized that there is an age‐related selective reduction in mitochondrial volume density and SDHmax per mitochondrial volume in type IIx/IIb DIAm fibers. In 24 Fischer 344 rats (6‐mo: n=6 female, n=6 male; 24‐mo: n=6 female, n=6 male), DIAm samples were dissected from the mid‐costal region and rapidly frozen at optimal sarcomere length. In 10 mm cross‐sections, DIAm fiber types were determined by immunoreactivity to specific myosin heavy chain (MyHC) isoform antibodies. Mitochondria were labelled using Mitotracker Green and imaged using 3D confocal microscopy (40X oil immersion lens; NA 1.4) with an optical slice of 0.5 µm. Each optical slice was deconvolved to increase spatial resolution (0.125 µm) and after thresholding, binarized images were reconstructed in 3D and analyzed for mitochondrial volume and mitochondrial complexity index (MCI=SA3/16µ2V2) using ImageJ and NIS elements. In alternate 6 mm sections, SDHmax was measured using a quantitative histochemical assay in which accumulation of nitro blue tetrazolium diformazan (NBTdfz) was measured in DIAm fibers every 15 s for 10 min. Consistent with previous findings, type IIx/IIb DIAm fibers were ~36% smaller in 24‐mo compared to 6‐mo rats (P<0.0001), whereas there were no age‐related differences in cross‐sectional areas (CSA) of type I and IIa fibers. Mitochondrial volume density was lower in IIx/IIb fibers of 24‐mo compared to 6‐mo rats (P<0.0001), whereas there were no age‐related differences in type I and IIa fibers. The MCI in type IIx/IIb fibers was lower (greater fragmentation) in 24‐mo compared to 6‐mo rats (P<0.0001), whereas the MCIs of type I and IIa fibers were similar across ages. Finally, SDHmax per fiber volume was lower in type IIx/IIb DIAm fibers in 24‐mo compared with 6‐mo rats (P<0.0001), whereas there were no age‐related differences in type I and IIa fibers. When SDHmax was normalized for mitochondrial volume, there was no age‐related difference in the intrinsic mitochondrial respiratory capacity in type IIx/IIb fibers. Therefore, aging results in reduced mitochondrial volume density and increased fragmentation in type IIx/IIb DIAm fibers, but no change in the intrinsic respiratory capacity of mitochondria.
An experimental animal model of cerebral aspergillosis
Authors: Sullivan Brianne , Baggett Mia A. , Guillory Christa , Jones Mary Jane , Steele Claude
Published: May 2022

Invasive pulmonary aspergillosis (IPA), a leading cause of death in immunosuppressed patients, is an infection most often attributed to the opportunistic fungi Aspergillus fumigatus (AF). The most common site of primary infection with this invasive disease is the lung, and the most prevalent site of secondary infection following hematological dissemination is the central nervous system (CNS). Dissemination of AF to the CNS, termed cerebral aspergillosis (CA), occurs in up to 40% of immunocompromised patients with IPA and has a mortality rate approaching 90%. While the experimental murine model of IPA and the resulting immune response have been extensively characterized, the current models available for CA are inconsistent and physiologically irrelevant. The lack of relevant models results in a considerable gap in knowledge for the neurological pathogenicity of infection. To address this gap, we infected wild‐type male and female C57BL/6 immunocompetent, or immunosuppressed (via high‐dose corticosteroid) mice using a one‐ (intranasal) or two‐hit (intranasal plus intravenous) method of inoculation with AF. Following immunosuppression and infection, mice were monitored for severe weight loss (>20%), neurological symptoms (e.g., altered‐gate, severe head‐tilt, and/or spinning in cage), and mortality daily for 10 days. At day 4 post‐inoculation, both brain and lung were assessed for fungal burden, and the immunological response to AF was evaluated in the brain. We observed significant weight loss, worse disease severity, and increased fungal burden in the brain in immunosuppressed mice infected via the two‐hit method. Further, in these mice, we found a significant disruption in cytokine and chemokine response in the brain as well as immune cell trafficking to the brain. In conclusion, we have developed a well‐defined, physiologically relevant, model of disseminated CA in immunosuppressed mice with primary pulmonary infection. This model will serve to advance understanding of the disease mechanism, identify immunopathogenic processes, and aid in defining the neuroinflammatory response to CA.
Effects of spliceosomal mutations on brain patterning and morphogenesis
Authors: Dong Yanchen , Beauchamp Marie‐Claude , Alam Sabrina , Bareke Eric , Majewski Jacek , Jerome‐Majewska Loydie
Published: May 2022

The major spliceosome consists of U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein (snRNPs), and each snRNP has distinct and sequential roles during the mRNA splicing process. Mutations in the core components of the spliceosome are associated with brain and neurological defects. The goal of this study is to examine the role of three core spliceosomal components, SNRPB, SF3B4, and EFTUD2, in brain patterning and morphogenesis, and whether or not they are required in the neural tube or neural crest cells for brain development. Previously, we identified brain defects associated with the mutation of these spliceosomal components in the neural tube and neural crest cells. Structures derived from the midbrain and hindbrain region, such as the diencephalon, pons, and cerebellum, were found to be absent in E12.5 and E14.5 mutant embryos. In this study, we aim to use mutant mouse models to identify shared transcripts and pathways disrupted by mutations in these spliceosomal genes. Through RNA‐seq analysis, we identified a number of mis‐expressed RNA‐binding proteins that may contribute to the malformationis found in these mutants. We plan to examine level and expression of these RNA binding proteins in wild type and mutant embryos to evaluate how they contribute to the phenotypes found. Furthermore, to assess the potential clinical application of our findings, we will use human embryonic stem cells (hESC) to generate human neural crest cells with mutatioin in these splicing factors; shared transcripts and pathways identified in the mouse model will be compared to those found in induced human neural crest cells. Distinct spliceosomophathies are difficult to diagnose due to phenotypic overlaps. The significance of this work lies in its potential to identify a disrupted pathway shared by mutations in various splicing factors, a therapeutic alternative across multiple spliceosomopathies.
Effects of Maximal Exercise on Central and Peripheral Arterial Stiffness in Adults with and without Intellectual and Developmental Disabilities
Authors: Marôco João L. , Angarten Vitor , Pinto Rita , Santos Vanessa , Santa‐Clara Helena , Fernhall Bo , Melo Xavier
Published: May 2022

Adults with Down‐Syndrome seem to be protected from early aging‐associated increases in arterial stiffness and to present blunted responses to maximal aerobic exercise in comparison to age‐matched controls, possibly underlying diminished vascular reserve. However, whether these arterial stiffness responses apply to adults with intellectual and developmental disabilities (IDD) in general remains unknown. We compared central and peripheral arterial stiffness response patterns between persons with and without IDD of different age groups following maximal aerobic exercise. We hypothesized that adults with IDD would show an attenuated arterial stiffness response to maximal aerobic exercise in comparison to young and middle‐aged adults without IDD. Thirty young adults with (n=15, age= 30 ± 7 years) and without (n=15, age= 27 ± 7 years) IDD, and 15 middle‐aged adults (age= 51 ± 4 years) without IDD performed a bout of maximal aerobic exercise. Central and peripheral arterial stiffness were measured at rest and following maximal aerobic exercise using applanation tonometry estimates of carotid‐femoral (cfPWV), carotid‐distal (cdPWV), and carotid‐radial (crPWV) pulse wave velocity, respectively. cfPWV was unchanged following maximal aerobic exercise in young adults with and without IDD (figure) but increased in middle‐aged adults (d= 0.85; 95% CI: 0.27 to 1.42 m.s‐1, p= 0.005), whereas cdPWV was reduced (d= ‐0.77; 95% CI: ‐1.06 to ‐0.48 m.s‐1, p< 0.001) and crPWV unchanged in all three groups, independent of changes in mean arterial pressure. Overall group differences suggest that young adults with (d= ‐1.78; 95% CI: ‐3.20 to ‐0.37 m.s‐1, p= 0.009) and without(d= ‐1.84; 95% CI: ‐3.26 to ‐0.43 m.s‐1, p= 0.007) IDD had lower cfPWV than middle‐aged adults. We found no evidence of early vascular aging and or a diminished vascular reserve following maximal aerobic exercise in adults with IDD.
Meditopia: Meditope‐Enabled Bonds and their Affinity with Cetuximab
Authors: Coler Zoe , Demars Stella , Faulkner Claire , Sureshkumar Sneha , Lane Jim
Published: May 2022

A meditope is a peptide found in the fragment antigen‐binding (Fab) region of an antibody, and can be used to bind substances, such as monoclonal antibodies (mAbs) and pharmaceuticals. We centered on four mutations that can increase the affinity between the meditope and the mAb, allowing for a stronger bond. Our focus was on the anti‐epidermal growth factor receptor mAb cetuximab, which tracks the overexpression of the epidermal growth factor receptor (EGFR) by cancer cells. Utilizing a meditope, cetuximab can be linked to chemotherapy, creating a more effective treatment that only attacks cancer cells, significantly reducing possible long‐term side effects. In this study, yeast display and deep mutational scanning tracked, enhanced, and joined four mutations within the meditope, increasing affinity, or bond strength, for cetuximab 10‐fold, from Kd 130 nm to 15 nm. Those mutations led to heightened cetuximab recruitment to EGFR‐overexpressing cancer cells, and therefore more efficient targeting of cancer. The methods used in this study can develop other effective meditope‐linked monoclonal antibodies and chemotherapy treatments. The Mahtomedi MSOE Center for BioMolecular Modeling MAPS Team will use 3‐D modeling and printing technology to examine structure‐function relationships of meditope‐mAb binding within cetuximab. The visual model will be a valuable tool in developing our story.
Proximal Tubule‐Specific Deletion of Angiotensin II Type 1a Receptors Augments Natriuretic Response to Atrial Natriuretic Peptide via NPRA/cGMP/NO Signaling Pathways in the Kidney
Authors: Wang Chih‐Hong , Li Xiao C. , Hassan Rumana , Sato Akemi , Dugas Courtney , Leite Ana P. O. , Sato Ryosuke , Zhuo Jia L.
Published: May 2022

We and others have previously shown that the proximal tubules of the kidney expressed high levels of angiotensin II (Ang II) type 1 (AT1) receptors and atrial natriuretic peptide (ANP) type A receptors (NPRA). In the proximal tubules, Ang II binds and activate AT1 (AT1a) receptors to stimulate proximal tubule Na+reabsorption, whereas ANP binds and activates NPRA receptors to inhibit Ang II‐induced proximal tubule Na+reabsorption. Interactions between Ang II and ANP systems play important counteracting roles in the proximal tubules to control proximal tubule Na+ reabsorption in the kidney and maintain blood pressure homeostasis. It is not known, however, whether the hypotensive and natriuretic responses to ANP are altered by the absence of AT1 (AT1a) receptor‐mediated antinatriuretic action of Ang II in the proximal tubules. The present study used a novel mouse model with proximal tubule‐specific knockout of AT1a receptors in the kidney, PT‐Agtr1a−/−, to test the hypothesis that deletion of AT1a receptors selectively in the proximal tubules augments the hypotensive and natriuretic responses to ANP. Two groups (n=9‐12 per group) of adult male wild‐type and PT‐Agtr1a−/− mice were infused without (time controls) or with ANP via osmotic minipump (10 ng/min, i.p.) continuously for 2 weeks. Basal blood pressure was about 16 ± 3 mmHg lower (P<0.01), fractional proximal tubule Na+ reabsorption was lower (P<0.05), and 24 h urinary Na+ excretion was higher in PT‐Agtr1a−/− mice (P<0.01), compared with wild‐type mice. ANP infusion decreased blood pressure and increased the natriuretic response in PT‐Agtr1a−/− mice further by inhibiting proximal tubule Na+ reabsorption, compared with wild‐type controls (P<0.01). The augmented hypotensive and natriuretic responses to ANP infusion in PT‐Agtr1a−/− mice was associated with increased plasma and kidney cGMP levels (P<0.01), kidney NPRA and NPRC expression (P<0.05), total eNOS and phosphorylated eNOS proteins (P<0.01), and urinary NO excretion (P<0.01), respectively. Taken together, the results of the present study provide further new insights into the important physiological roles for intratubular Ang II/AT1a and ANP/NPRA in the proximal tubules to regulate proximal tubule Na+ reabsorption and help maintain blood pressure homeostasis.
Automated, Score‐Based Pharmacophore Generation Using Multiple Copy Simultaneous Search
Authors: Szwabowski Gregory L. , Parrill Abby L. , Baker Daniel L.
Published: May 2022

Pharmacophores are three‐dimensional arrangements of molecular features required for biological activity that are often used in virtual screening efforts to prioritize ligands for experimental screening. G protein‐coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for ligand discovery and drug development. Pharmacophore models are most typically ligand‐based and constructed via the identification of structural commonalities between known bioactive ligands. However, structure‐based pharmacophore models (requiring only a target protein’s structure) provide an alternative to ligand‐based pharmacophore models. Although both ligand‐based and structure‐based pharmacophore models have exhibited success in prior virtual screening studies, most pharmacophore modeling efforts are not applicable to GPCR targets lacking known ligands. Consequently, the development of a purely structure‐based pharmacophore modeling protocol for GPCR has become an increasingly attractive approach as the number of publicly available, high‐resolution GPCR structures has increased (113 unique GPCR represented as of November 24, 2021). Thus, the aim of this work was to develop a structure‐based pharmacophore modeling approach that can generate well‐performing pharmacophore models in GPCR crystal structures and homology models. Pharmacophore models were generated in crystal structures and homology models of 13 class A GPCR via automated feature annotation of differing subsets of functional group fragments placed with Multiple Copy Simultaneous Search (MCSS). During the feature annotation process, distance cutoffs were enforced to generate combinations of pharmacophore features that reflect spatial arrangements of interactions typically observed in GPCR pharmacophore models. Resulting pharmacophore models for each target GPCR were used to search a database containing active and decoy/inactive compounds for 30 class A GPCR and scored using enrichment factor (EF) and goodness‐of‐hit (GH) metrics to assess performance. To identify well‐performing pharmacophore models for cases where active ligands are unknown, pharmacophore models were classified using cluster‐then‐predict logistic regression. As a proof‐of principle for this method, pharmacophore models were generated for the orphan GPCR GPR101 to elucidate candidate ligands that may possess activity for GPR101. Application of this method to the set of 13 class A GPCR targets resulted in the generation of pharmacophore models possessing EF scores ≥ 2 in both crystal structures (12 of 13 cases) and homology models (9 of 13 cases). In addition, classification of pharmacophore models with cluster‐then‐predict logistic regression resulted in positive predictive values (PPV) of at least 0.67 for pharmacophore models generated with selected MCSS fragment subsets. Lastly, implementation of generated pharmacophore models in a virtual screening workflow identified 33 candidate ligands for GPR101.
Structural and biochemical analysis of phosphoethanolamine methyltransferase from the pine wilt nematode Bursaphelenchus xylophilus
Authors: Lee Soon Goo
Published: May 2022

In free‐living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x‐ray crystal structure of the enzyme as a dead‐end complex with phosphoethanolamine and S‐adenosylhomocysteine. The three‐dimensional structure of BxPMT1 served as a template for site‐directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady‐state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the β1d‐α6 loop (W123F, M126I, and Y127F) and β1e‐α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
Investigating Substrate Specificity For 6‐Hydroxynicotinate 3‐Monooxygenase (NicC) With Coumalic Acid: Consequences To Catalysis Of Replacing or Removing The Ring Nitrogen
Authors: Hlaing May Z. , Campbell Ryan , Snider Mark J.
Published: May 2022

6‐Hydroxynicotinate 3‐monooxygenase (NicC) is a group A flavin dependent monooxygenase (FDM) that catalyzes the regioselective decarboxylative hydroxylation of 6‐HNA into 2,5‐dihydroxypyridine (DHP) with the concomitant oxidation of NADH to NAD+. This enzyme‐catalyzed reaction involves three segments: (1) substrate binding, (2) FAD reduction by NADH, and (3) hydroxylation‐decarboxylation of 6‐HNA through a hydroperoxy‐flavin intermediate. Past studies have shown that NicC can catalyze this reaction with alternate aromatic substrates including 4‐hydroxybenzoic acid (4‐HBA), a substrate analogue with a homocyclic ring (i.e. lacking the ring nitrogen). However, 4‐HBA is bound weakly (355 ± 66 μM) and only 50% of the reaction progresses to substrate hydroxylation, versus hydroperoxy‐flavin degradation to H2O2, suggesting the importance of the ring nitrogen in the coupling efficiency of NicC. To further explore the role of the ring nitrogen, the efficiency of the reaction with coumalic acid (CA) was determined. The NicC‐CA dissociation constant measured by monitoring the change in absorbance at 450nm (flavin) shows that NicC binds CA (Kd = 5 ± 1 μM) 10x more tightly than 6‐HNA (Kd = 58 ± 12 μM). Binding of CA by NicC is an isothermal process (∆H = 0), indicating that it is driven by a favorable change in entropy. Analysis of the reaction products by Q‐TOF MS indicates that NicC catalyzes the decarboxylative hydroxylation of CA into 5‐hydroxy‐2H‐pyrane‐2‐one. However, coupling experiments by HPLC analysis and detection of H2O2 by a fluorescence assay both indicate that the reaction with CA is highly uncoupled with only minor 5‐hydroxy‐2H‐pyrane‐2‐one formation. Steady‐state kinetic analyses show that CA bound to NicC enhances its rate of NADH oxidation 16‐fold (by comparison, 6‐HNA enhances the rate of NicC NADH oxidation 30‐fold). Transient state kinetic analyses by stopped flow under anaerobic conditions to permit single‐turnover kinetics shows that, unlike in the binding of 6‐HNA, the binding of CA does not enable the formation of a charge‐transfer complex prior to FAD reduction. These results indicate that an H‐bond accepting group in the aromatic ring at position 1 is important for establishing the correct conformation of the ES complex to enable effective binding, flavin reduction and, ultimately, substrate hydroxylation.
Mild‐to‐Moderate Chronic Hypercapnia Impairs Adaptation of Acute CO2/H+ Chemosensitivity but Not Steady‐State Ventilation
Authors: Buchholz Kirstyn J. , Burgraff Nicholas , Neumueller Suzanne , Pan Lawrence , Hodges Matthew , Forster Hubert
Published: May 2022

CO2 retention (hypercapnia, CH) presents in a number of etiologies including sedative use, chronic lung disease, and central/peripheral neuromuscular disorders. Acute‐on‐chronic hypercapnia can develop in these disorders during times of acute stress (i.e., infection and postoperative states). Recurrent acute‐on‐chronic CH may lead to respiratory failure and transient, life‐threatening worsening of the hypercapnia. We previously established in awake goats that exposure to mild CH (PaCO2 ~55mmHg) elicits changes across multiple physiologic systems but does not impair the response to acute increases in inspired CO2(InCO2).Herein, we test the hypothesis that progressive exposure from mild‐to‐moderate CH (~55‐to‐65mmHg CO2) results in secondary adaptations across several physiologic systems but increases the risk of pathological responses during acute‐on‐chronic exacerbations. Utilizing our established large animal model of CH, female goats (n=7) were exposed to 7 days (d) of 6% InCO2 followed by 7d of 8% InCO2. Throughout the protocol, we regularly assessed steady‐state cardiorespiratory function (i.e., VI, f, VT, HR, and BP) and acute CO2/H+ chemosensitivity. During 6% InCO2, the temporal pattern of physiologic adaptations was consistent with that previously reported (J. Physiol. 2018). As expected, increasing steady‐state (SS) InCO2 resulted in a sustained 10 mmHg increase in PaCO2 and an increase in arterial [H+] that decreased over the 7d at 6%. During 6% InCO2 there was a transient decrease (6%‐Day 2) in acute chemosensitivity (DVI/DCO2) that returned to control values by Day 5 at 6%. Upon increasing SS InCO2from 6% to 8%, minute ventilation (VI) increased from 23±3.4L/min on Day 7 at 6% InCO2 to 34±2.2L/min on Day 1 at 8% InCO2,which was primarily driven by an increase in breathing frequency. By 24h at 8% InCO2, VIhad declined from the 8%‐Day 1 values and remained at or near that level thereafter. This temporal pattern of adaptation is comparable to that observed during 6% CO2. A similar pattern of adaptation in [H+] occurred during 8% as occurred at 6%, while [HCO3‐] continually increased. PaCO2remained elevated throughout the 8% InCO2 exposure, compared to 6% InCO2. Finally, when SS InCO2 was increased to 8% the acute chemoreflex remained suppressed throughout the 7d. Our results suggest that progressive worsening of chronic hypercapnia results in similar SS adaptations compared to initial hypercapnic exposure. However, more severe levels of CH, such as 8% InCO2, perturb the acute chemoreflex, creating a risk for consequences such as CO2 narcosis and cardiorespiratory failure in patients with pre‐existing CH.
Palmitoylation of RGS20 Affects its Cellular Function
Authors: Zhang Qian , Sjogren Benita
Published: May 2022

Regulator of G protein signaling 20 (RGS20) is a member of the RZ family of the RGS protein superfamily, which serve as key negative regulators of G protein‐mediated signal transduction. Previous studies showed that overexpression of RGS20 is correlated with progression of triple‐negative breast cancer (TNBC) in tumor tissues and contributes to breast cancer cell migration and invasion. However, there is still a large gap relating to the regulation and function of RGS20 due to its late discovery. RGS20 contains a poly cysteine string motif, which is assumed to be heavily palmitoylated. Palmitoylation, an important post‐translational protein modification, plays an important role in cells by changing cellular functions of proteins. Consequently, in this study we determined palmitoylation regulation of RGS20, which could provide more information for new potential treatment strategies for TNBC. We used click chemistry to show that RGS20 is palmitoylated in transfected HEK‐293T cells and MDA‐MB‐231 triple negative breast cancer cells. We also showed that constitutively activated Gαo, GαoQ205L, significantly increases palmitoylation levels of RGS20. Furthermore, palmitoylated RGS20 associates much more with GαoQ205L, while de‐palmitoylated RGS20 lost the association with GαoQ205L. Next, we detected how palmitoylation affects RGS20 GAP activity, as well as cAMP signaling downstream of Gαo. We mapped the palmitoylation site crucial for all of these effects to a conserved cysteine in the RGS domain. Mutating this cysteine residue resulted in a significant decrease in both palmitoylation signal and the association with GαoQ205L. Overall, we showed that palmitoylation affects functions of RGS20 in cells and that a conserved cysteine in the RGS domain is a critical site for its palmitoylation.
Obeticholic Acid Prevents Fibrosis in a Model of Tubulointerstitial Kidney Disease
Authors: Davidson Shania R. , Adapa Sharmila , Lopez‐Santiago Isabel , Dial Katelyn , Rowland Emma , Mykala Komuraiah , Wang Xiaoxin , Rosenberg Avi , Levi Moshe , Jones Bryce A.
Published: May 2022

Objective

This study investigates the protective properties of farsenoid X receptor (FXR) agonism by the drug Obeticholic Acid (OCA) in the adenine‐induced tubulointerstitial fibrosis model of kidney disease male and female mice.

Hypothesis

OCA treatment will decrease the expression of SMAD3 and phosphorylated‐SMAD3, and therefore will be nephroprotective in the adenine model of kidney disease in male and female mice.

Methods

Male and female C57BL/6J mice (12 weeks old) were fed chow (Research Diets D1912040li) or chow admixed with adenine (0.2% w/w) ad lib for 7 weeks. Mice were treated with either vehicle (corn oil) or obeticholic acid (10 mg/kg BW) by gavage 5 days per week. Plasma and organs were collected and processed for bio‐chemical analysis.

Results

OCA treatment reduced BUN and plasma creatinine in diseased mice of both sexes. FXR agonism reduced SMAD3 expression in both male (P<0.05) and female (P<0.05) mice. Importantly, FXR agonism also reduced SMAD3 phosphorylation in both male (P<0.05) and female (P<0.001) mice. On histology, OCA reduced renal fibrosis in male (P<0.05) but not female mice.

Conclusions

FXR agonism by OCA is anti‐fibrotic in the adenine model of tubulointerstitial fibrotic crystalline nephropathy in male and female mice. Mechanistically, OCA prevents renal fibrosis by reducing SMAD3 expression and phosphorylation.