bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformationally altered hyaluronan mitigates the symptoms of Parkinson disease in mice
Authors: Sun Tzu‐Yu , Lee Kuan‐Ting , Chang Nan‐Shan
Published: May 2022

Native hyaluronan (HAn) is poor in exerting anti‐inflammatory responses. However, interchain‐dissociated or conformationally altered HA significantly inhibits complement activation. To induce interchain dissociation, HA was heat treated, quickly frozen, and quickly thawed prior to interacting with serum complement. Here, HAn is subjected to sonication for indicated times. Heat was generated up to 70oC during sonication. Time‐related sonication HA preparations (HAson) exhibit various extent of degradation. These samples are conformationally altered due to heat treatment for 1 to 24 hr and the resulting degradation. We examined whether HAn and HAson block rotenone‐induced Parkinson’s disease (PD) in mice. Rotenone, an environmental pesticide, inhibits mitochondrial complex I and causes aggregation of α‐synuclein for leading to neuronal death. Immune competent BALB/c and C57BL/6 mice received PBS, HAn, or HAson preparations (200 μg in 100 µl PBS) via tail vein injections for two consecutive weeks, and were allowed to rest for 14 days. The mice were then challenged with rotenone (5 mg/kg) via subcutaneous injections and subjected to indicated tests two weeks later. Mice receiving HAson8 (sonication for 8 hours) significantly resisted the development of rotenone‐induced acute PD symptoms (~80% of improved mouse mobility by footprint analysis), as compared to mice receiving PBS, HAn, and other HAson preparations (4, 12, 16 and 24 hr sonication), respectively. HAson8 also blocked pentylenetetrazol‐mediated seizure in mice. All the HA preparations were not toxic to the animals, and did not alter the normal behavior of mice during experiments involving rotarod test and Barnes water maze analysis. As a potent inhibitor of Alzheimer’s disease, Zfra4‐10 peptide significantly restores memory loss, reduces PTZ‐mediated seizure and retards PD progression. Mechanistically, Zfra4‐10 binds membrane hyaluronidase Hyal‐2 to signal with WWOX and Smad4 to activate Hyal‐2+ spleen Z cells for blocking neurodegeneration. It is suggested that compared to HAn, HAson8 strongly binds membrane Hyal‐2 for Z cell activation and mitigating PD symptoms.
Norepinephrine mediates effects of chronic amphetamine on memory performance in aged rats
Authors: Scognamiglio Serena , Dezfuli Ghazaul , Kellar Kenneth
Published: May 2022

Understanding the neural mechanisms of normal and pathological aging is increasingly important as the average lifespan continues to rise. Memory impairment is attributed to normal aging processes, with the hippocampus and the cerebral cortex playing key roles in the encoding and retrieval of memories. Norepinephrine (NE) is an important modulator of memory, especially emotional memory (Tully et al., 2010). Previously, we demonstrated that acute treatment with amphetamine (0.5mg/kg) enhances memory performance on the novel object recognition (NOR) task in aged Fischer 344 rats, with a reversible and short‐term effect (Scognamiglio et al., 2021). The purpose of this study was to investigate whether chronic amphetamine treatment has a more robust and possible long‐term effect on memory performance in aged rats and whether the effect of amphetamine on object recognition memory is NE‐ dependent. To this end, in the first set of experiments we administered amphetamine for 2 weeks via drinking water at doses of 0.25 mg/kg (week 1) and 0.5 mg/kg (week 2) and tested aged Fischer 344 rats on the NOR task. To determine the extent to which NE influences the cognitive effects of amphetamine, we examined the effects of systemic administration of the β‐ and α‐adrenergic antagonists propranolol, metoprolol, and prazosin prior to the NOR test phase in aged rats chronically treated with amphetamine. The present studies found: (a) aged rats chronically treated with amphetamine displayed improved object recognition memory compared to rats treated acutely, (b) memory performance returned to baseline 2 months after chronic amphetamine treatment, (c)all three of the β‐ and α‐ adrenergic antagonists abolished the effect of amphetamine on memory performance in the NOR test. An additional goal of this study was to assess the outcome of chronic amphetamine treatment on a cellular level by analyzing dendritic spines in the cerebral cortex and hippocampus. The results of those studies will be reported.
Endothelial‐specific nanodelivery of Rho kinase inhibitors: Targeting tumor angiogenesis and metastasis
Authors: Lahooti Behnaz , Akwii Racheal , Mattheolabakis George , Mikelis Constantinos M.
Published: May 2022

Objective

The limitations of current vascular endothelial growth factor (VEGF)‐centered anti‐angiogenic therapy include the inhibition of physiological angiogenesis, development of evasive resistance, and increased vascular leakage, which drives the need for a novel therapy for tumor angiogenesis and metastasis. RhoA pathway inhibition blocks VEGF‐ and sphingosine‐1 phosphate (S1P)‐induced angiogenesis and vascular leakage without hindering endogenous angiogenesis. Fasudil, a clinically relevant Rho‐associated kinase (ROCK) inhibitor, blocks the RhoA downstream signaling, but adverse events (hypotension, skin reactions) exist. Here, we propose endothelial ROCK targeting with Fasudil‐containing immune‐related nanodelivery.

Hypothesis

Fasudil‐encapsulated CD31‐targeting liposomal formulation is a potential tumor antiangiogenic therapy by blocking RhoA signaling via ROCK inhibition in the endothelial cells.

Methods

Fasudil‐liposomal nanoparticles were prepared by DPPC, cholesterol, 16:0 PEG2000PE, DSPE‐PEG2000‐mal, DSPE‐PEG‐FITC, and CHCl3: MeOH (4:1) combination, thin‐film formation, downsizing after Fasudil incorporation, filtering, and conjugation with IgG and CD31‐targeting antibodies. We used dynamic light scattering (DLS) for the liposomal characterization, dose‐response experiments on primary Human Umbilical Vein Endothelial Cells (HUVEC) and Bend3 cells for in vitro toxicity evaluation, antibody (Ab)‐to‐liposome binding evaluation and pathway inhibition evaluation by western blot, and UPLC analysis (EE)% for the evaluation of encapsulation efficiency. The binding affinity of the CD31‐conjugated liposomes and their time‐dependent cell incorporation were evaluated by competitive binding assay via immunohistochemistry (IHC) and flow cytometry. Morphometric analysis was assessed by Livecyte imaging analysis.

Results

Size and PDI measurement by DLS confirmed the stability of our formulation, which presented limited toxicity (concentration: 0.625‐5120 µM; IC50 at 43.05 µM [HUVECs over 24 hours of treatment]) with a significantly higher overall survival rate than Fasudil. Efficient CD31 and mouse IgG Ab conjugation to Fasudil liposomes and blockade of the RhoA downstream signaling in endothelial cells were confirmed. The specific binding of CD31‐targeting liposomes on endothelial cells was verified in the presence and absence of excess CD31 Ab. The time‐dependent study confirmed the kinetics of liposome incorporation in endothelial cells and the enhanced uptake of CD31 liposomes compared to IgG. Cell morphometric and physiological analysis revealed comparable alterations caused by Fasudil‐ and endothelial‐specific Fasudil‐containing liposomes.

Conclusion

We have successfully developed Fasudil‐encapsulated, CD31‐targeting liposomes. Future studies will be directed to study the formulation’s effect on endothelial physiology in vitro and in vivo, tumor growth, angiogenesis, and metastasis.
Histological Diversity And Evolution Of Lizard Osteoderms
Authors: Vickaryous Matthew , Williams Catherine , Willan Gabriella , Kirby Alex , Herrel Anthony , Kever Loic , Moazen Mehran , Marghoub Arsalan , Rai Shreya , Abzhanov Arkhat , Evans Susan
Published: May 2022

Many reptiles reinforce the dermis with discrete mineralized organs known as osteoderms. Among lizards, osteoderms demonstrate species‐specific differences in size, shape, and distribution across the body. Whether osteoderms also vary in details of tissue composition, including the organization of the fibrillar matrix, remains unclear. Here, we investigate osteoderm histology in three species‐rich lizard groups: gekkotans (geckos), scincids (skinks), and anguimorphans (anguids, helodermatids, and related taxa). With one possible exception, all lizard osteoderms are dominated by bone tissue. Unexpectedly, representative members of all the major groups develop an enigmatic, collagen‐poor capping tissue. Gekkotan osteoderms are rare (<2% of species) and demonstrate considerable histological heterogeneity between species. This includes variation in bone matrices, and the development of additional (non‐osseous) skeletal tissues. In the gecko genus Geckolepis, putative osteoderms appear to lack bone and instead are composed of dense collagen plates capped by a vitreous collagen‐poor tissue. Unlike geckos, osteoderms are common to virtually all skinks. Most skink osteoderms are compound elements, composed of multiple conjoined smaller plates (referred to as osteodermites). Histologically, skink osteoderms are dominated by lamellar bone with well‐organized Sharpey’s fibres linking adjacent plates together and some woven and parallel‐fibred bone. Most species develop the collagen‐poor capping tissue, particularly where adjacent osteodermites articulate with one another. Osteoderms are also common to many anguimorphans, but the histology varies considerably between taxa. While anguid and helodermatid osteoderms demonstrate multiple bone matrices (woven‐fibred, parallel‐fibred, lamellar bone, and Sharpey‐fibred), Shinisaurus osteoderms are primarily woven‐fibred and Sharpey‐fibred bone, and those of Varanus are mostly parallel‐fibred bone. Expression of the capping tissue also varies, from thick and well‐defined (Heloderma) to discontinuous (e.g., several anguids) or entirely absent (Shinisaurus, Varanus). We propose that the histological diversity observed in lizard osteoderms is driven by factors including phylogeny, ecology, and function. The identity of the capping tissue remains unresolved, but our findings reveal it is far more widespread than previously considered. The results presented here provide important steps towards understanding the diversity and evolution of the skeleton in the skin.
Analyzing the 5′‐3′ Kissing‐Loop Interaction Sequence for Cap‐Independent Translation Initiation of Blackcurrant Reversion Virus
Authors: Zhou Liheng , Lee Julie , Baquero‐Galvis Laura D. , Filbin Megan E.
Published: May 2022

Translation regulation commonly involves the 5′‐m7GpppN cap which binds the initiation factor 4E bound to recruit the other initiation factors and the small ribosomal subunit. Yet some viruses, such as the Blackcurrant Reversion virus (BRV), have no 5′‐cap but are still able to translate using alternative mechanisms such as 3′ cap independent translation enhancers (3′ CITE). The 3′ CITEs in BRV’s bipartite genome are predicted to work like 4E by recruiting initiation factors and bringing them to the 5′ end for initiation of translation though a 5′‐3′ RNA kissing interaction, called the kissing‐loop interaction sequence (KIS). Our goal is to verify whether or not the KIS between the 5′ UTR and 3′ CITE exists to promote translation initiation. Using site‐directed mutagenesis and in vitro transcription, we created a series of KIS mutations (including compensatory mutations). Our preliminary in vitro luciferase translation experiments indicate the putative KIS between the 5′ UTR and 3′ CITE may not be important for translation initiation, however, we are currently working to verify these results and identify alternative KISs between the 5′ and 3′ ends. Although many CITEs use 5′‐3′ RNA kissing interactions to deliver translation machinery to the 5′ end of the genome, it is not required for CITE function. The BRV CITE may function through a protein bridge or some other unknown mechanism.
Structural Investigation of a Putative Intrinsically Disordered Region Within Deleted in Colorectal Carcinoma That Regulates Protein Synthesis
Authors: Nowling Nathan L. , Spear Elizabeth A. , Paukovich Natasia , Manalastas John , Vögeli Beat , Filbin Megan E.
Published: May 2022

Intrinsically disordered proteins (IDPs) are key players in cell signaling, gene expression networks, and cellular development – to include spatiotemporally‐controlled protein synthesis. The intracellular, C‐terminal tail of the chemotropic receptor, Deleted in Colorectal Carcinoma (DCC), is a predicted IDP that stalls translation machinery at axon growth‐cone membranes. DCC releases the translation machinery (via an unknown mechanism) when it encounters its specific ligand Netrin‐1, initiating local protein synthesis and axonal outgrowth. Using mutational analysis coupled with in vitro translation reporter assays, we have identified a portion of DCC’s C‐terminal tail that is important for translation stalling. To determine any structure and dynamics of this putative IDP, we are recombinantly expressing and purifying the protein for analysis via NMR. Our analysis will shed light on how protein structural plasticity and dynamics play a role in regulating important cellular processes and may provide avenues for predictive modeling and target identification of IDPs that regulate translation.
Loss of PTPN2 Activity Alters Iron Handling Protein Expression in IBD Patients and Causes Iron Deficiency in Mice
Authors: Lei Hillmin , Shawki Ali , Spalinger Marianne R. , Canale Vinicius , Santos Alina N. , Chatterjee Pritha , Crawford Meli’sa S. , Manz Salomon , Becerra Anica , Scharl Michael , McCole Declan F.
Published: May 2022

Background

Anemia is the most common extraintestinal complication of inflammatory bowel disease (IBD) and is a risk factor for Crohn’s disease (CD) onset. Iron deficiency is the most common cause of anemia in IBD; however, the mechanisms involved are poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non‐receptor type 2 (PTPN2), in regulating iron homeostasis.

Methods

Proteomic analyses were performed on serum from IBD patients genotyped for the IBD‐associated loss‐of‐function rs1893217 PTPN2variant (n=10/genotype). Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) 3‐week‐old mice were analyzed for serum iron content, liver non‐heme iron and liver expression of the iron regulatory hormone, hepcidin (Hamp1). Protein and RNA from duodenal epithelial cells (DECs) were assayed by western blotting and qPCR. Localization of the brush border ferrous iron transporter, DMT1, in duodenal tissue was determined by immunohistochemistry (IHC).

Results

Iron homeostasis genes, the iron carrier transferrin (TF) and the transferrin receptor (TFRC), were reduced (‐log p‐value = 10.7) in CD patients with the PTPN2 risk variant. Ptpn2‐KO mice had reduced i) serum iron (p<0.001; n=11); ii) serum TF saturation (p<0.01; n=9); and iii) liver non‐heme iron levels (p<0.01; n=10), vs. Ptpn2‐WT and Het mice. This indicated that PTPN2 loss decreased serum and liver iron storage. Moreover, Ptpn2‐KO mice had reduced liver expression of Hamp1 (p<0.01; n=7), likely suppressed by low serum iron. DEC gene expression of ferritin (Fth1), an intracellular iron storage molecule, was reduced (p=0.048; n=5) while Tfrc1, a mediator of basolateral cellular iron uptake, was significantly increased (p=0.0048; n=6) in Ptpn2‐KO mice. Reduced FTH1 expression (p=0.007; n=12) in DECs of Ptpn2‐KO mice was confirmed by western blot, indicating reduced intracellular iron storage. DMT1 expression was unchanged (n=6), whereas IHC showed reduced apical membrane DMT1 in duodenal epithelium of Ptpn2‐KO mice (n=6), suggesting a possible mechanism of impaired intestinal non‐heme iron uptake.

Conclusions

CD patients with the PTPN2 loss‐of‐function rs1893217 SNP display alterations in serum iron handling proteins. Loss of PTPN2 activity in mice causes features of anemia including iron deficiency associated with mislocalization of the duodenal transporter DMT1. These findings identify a major role for PTPN2 in regulating iron homeostasis.
A Randomized Controlled Trial of Dietary Supplementation with Prunes (Dried Plums) on Inflammatory Markers in Postmenopausal Women
Authors: Damani Janhavi J. , Strock Nicole C. A. , De Souza Mary Jane , Rogers Connie J.
Published: May 2022

The prevalence of osteoporosis among women aged 50 years and older is expected to reach 13.6 million by 2030. Osteoporosis is characterized by compromised bone strength due to reduced bone mineral density (BMD) and bone quality, representing a major public health issue that necessitates effective treatment regimens that are safe, cost‐effective, and associated with fewer adverse effects than pharmaceutical agents. Alternative dietary interventions such as prunes (dried plum) have been extensively studied to mitigate bone loss in preclinical models of osteoporosis. In postmenopausal women, estrogen deficiency triggers an upregulation of inflammatory pathways, which promotes bone loss, thus increasing risk of fractures. Our understanding of the effect of prune consumption on inflammatory markers in humans is limited, warranting further investigation. This study aimed to evaluate the effects of 12 months of prune consumption (two doses) on inflammatory mediators. Postmenopausal women (n=106, 55‐75 years old) with low BMD were randomized to consume 0g prunes/day (control), 50g prunes/day, or 100g prunes/day for 12 months. All participants received 1200mg calcium and 800 IU vitamin D3 as standard of care. We hypothesized that prune consumption at 50g/day and 100g/day will reduce inflammatory markers in postmenopausal women with low BMD compared to control group (0g/day). At baseline and after 12 months of prune consumption, serum and peripheral blood mononuclear cells (PBMCs) were isolated to quantify C‐reactive protein (CRP) using a high‐sensitivity CRP immunoassay, the number of circulating monocytes using flow cytometry, and pro‐inflammatory cytokines [interleukin (IL)‐1β, IL‐6, IL‐8, monocyte chemoattractant protein (MCP)‐1, and tumor necrosis factor (TNF)‐α] from lipopolysaccharide (LPS)‐stimulated PBMCs using a multiplex enzyme‐linked immunosorbent assay. Differences among groups were assessed using one‐way ANOVA or Kruskal‐Wallis test. No significant differences in baseline characteristics were found among the three treatment groups. Prune consumption did not alter serum CRP or the number of monocytes. However, consumption of 100g prunes/day resulted in a significant reduction from baseline in IL‐1β (p=0.013), IL‐6 (p=0.007), and IL‐8 (p=0.049) secretion and consumption of 50g prunes/day resulted in a significant reduction from baseline in TNF‐α (p<0.001) secretion from LPS‐stimulated PBMCs compared to the control group. Our findings demonstrate that 12 months of prune consumption suppresses inflammatory cytokines from LPS‐stimulated PBMCs. Thus, daily consumption of 50g‐100g of prunes may reduce inflammatory mediators that can contribute to bone loss in postmenopausal women.
Microtubule Stabilizers as Modifiers of Alpha‐Synucleinopathy
Authors: Jamenis Anuj S. , Bhatia Tarun N. , Miner Kristin M. , Kaku Krishna , Shah Yesha , DeMarco Brett A. , Clark Rachel N. , Gangjee Aleem , Leak Rehana K.
Published: May 2022

Lewy body disorders are associated with hallmark inclusions containing aggregated forms of alpha‐synuclein. When alpha‐synuclein aggregates in the synapse, it may be transported along axonal microtubular tracks to the soma for degradation or deposition into insoluble inclusions. However, during this transportation, alpha‐synuclein aggregates may also adhere to the microtubules and impede the axonal transport of other cellular constituents. Axonal transport is known to depend upon the stability of the microtubule tracks, but the specific impact of alpha‐synucleinopathy on microtubular dynamicity is unclear. Here, we assessed markers of microtubule stabilization in olfactory bulb and amygdala samples harvested postmortem from men and women with Lewy body disorders and age‐matched control subjects. Unexpectedly, we observed that women showed higher levels of detyrosinated alpha‐tubulin (marker of microtubule stability) as well as higher levels of tyrosinated alpha‐tubulin (marker of non‐stabilized microtubules) compared to men. Second, the impact of microtubule stability on inclusion body formation is also poorly understood. Thus, we measured inclusion counts in primary hippocampal cultures treated with novel heterocyclic microtubule stabilizers and challenged with preformed alpha‐synuclein fibrils. As expected, the microtubule stabilizers reduced pathologically phosphorylated (pSer129) alpha‐synuclein+ inclusion counts, and the microtubule destabilizer nocodazole prevented this reduction in pathological inclusions. These collective data suggest that microtubule stability may influence the emergence of alpha‐synucleinopathy in experimental Lewy body disease.
Single‐Cell Reconstruction of Cellular Heterogeneity of Congenital Heart Disease
Authors: Tambi Richa , Nandkishore Sharon , Sharafat Shermin , Kader Faiza , Nassir Nasna , Binte Ashraf Zehra , Mohamed Nesrin , Ahmed Awab , Abdel Hameid Reem , Taylor Alan , Abou Tayoun Ahmad , Alsheikh‐Ali Alawi , Di Donato Roberto , Uddin Mohammed , Berdiev Bakhrom
Published: May 2022

Congenital heart disease (CHD) involves cardiovascular malformations and, are one of the most common type of birth defect. The estimated prevalence ranges from ~1‐3% of live births and it causes significant mortality worldwide. Although etiology and pathophysiology of CHD remains elusive, genetic factors are known to be a major contributor. In this study, we aim at deciphering the genomic, transcriptomic, and proteomic landscape of the mutations associated with CHD. We collected 14209 SNVs, and 63 CNVs representing a sample size of 3,408 individuals, from 58 different articles published between January 2000 and December 2020. We identified 6,641 genes impacted by these mutations of which the MYH6, GATA6, NOTCH1, OBSCN and PTPN11 harbour the most number of recurrent mutations. We observed a high prevalence of early onset and male subjects with CHD, carrying a mutation. Stringent downstream meta‐analysis will be performed on this preliminary dataset. We plan to first comprehensively annotate the collected mutations using ANNOVAR and GenomeArc Analytics. The mutations will be then classified on the basis of American College of Medical Genetics (ACMG) guidelines to retain clinically relevant (pathogenic, likely pathogenic and variant of unknown significance) rare mutations. Next, we will analyse the mutation hotspots using 3D protein structures to determine domains which are most affected by these mutations. Furthermore, we will investigate the spatiotemporal developmental (prenatal to adult) human heart single‐cell OMICs (transcriptome and proteome) datasets in order to identify CHD associated cell types. Our analysis will detect cell types where marker genes are enriched with clinically relevant mutations. Our study aims to uncover the genetic and cellular heterogeneity of CHD which will aid precision diagnosis and therapeutics.
Conditional deletion of the glucocorticoid receptor in vascular smooth muscle disrupts the circadian rhythm of vascular reactivity and reduces blood pressure
Authors: Krilis Georgios , Bailey Matthew A. , Ivy Jessica R.
Published: May 2022

Glucocorticoids synchronise peripheral clocks with the master clock in the suprachiasmatic nucleus in response to the ambient light cycle. In humans and mice, arrhythmic glucocorticoids induce non‐dipping blood pressure and vascular dysfunction. The mechanisms of this are poorly understood. We hypothesize that arrhythmic activation of the glucocorticoid receptor attenuates the circadian clock signalling in the vasculature leading to changes in blood pressure. To test this hypothesis, we assessed vascular function and blood pressure rhythms in control mice and mice with smooth muscle specific deletion of the glucocorticoid receptor (SMGRKO).

Renal and mesenteric arteries were isolated at 7am (ZT0; lights on) and 7pm (ZT12; lights off), mounted on a wire myograph and the responses to increasing doses of phenylephrine or sodium nitroprusside (SNP) assessed. In wild‐type mice, the sensitivity of the renal artery to phenylephrine‐mediated contraction was greater at ZT12 than at ZT0, by comparing the half maximal effective concentration of each group, (logEC50: 7.54±0.26 versus 7.29±0.17 respectively; p=0.04). The sensitivity to SNP‐mediated relaxation was also greater when measured at ZT12 compared to ZT0 (7.64±0.3 versus 7.09±0.5; p=0.03). Similar responses were also detected in the mesenteric arteries. In SMGRKO mice, the temporal differences in renal and mesenteric artery vascular reactivity were absent. We used radiotelemetry to assess the effect of this altered rhythm of vascular reactivity on blood pressure. SMGRKO had significantly lower systolic blood pressure throughout the 24h cycle compared to wild‐type littermates, both when lights were off (107.9 ± 22.13 versus 136.3 ± 17.17 mmHg respectively; p=0.015) and during the lights on period (99.6 ± 13.16 versus 118 ± 15.9 mmHg; p=0.014). Blood pressure rhythmicity was not altered in SMGRKO mice.

These data show that the glucocorticoid receptor signalling in vascular smooth muscle contributes both to the diurnal variation of vascular reactivity and to blood pressure, but loss of the receptor does not affect blood pressure rhythm. Whether glucocorticoid receptor deletion in smooth muscle protects against non‐dipping blood pressure induced by glucocorticoids needs further investigation.
Contributions of Cathepsin A and Carboxylesterase 1 to the hydrolysis of Tenofovir Alafenamide in the Human Liver, and the Effect of CES1 Genetic Variation on Tenofovir Alafenamide Hydrolysis
Authors: Li Jiapeng , Shi Jian , Zhu Haojie
Published: May 2022

The prodrug tenofovir alafenamide (TAF) is a first‐line antiviral agent for the treatment of chronic hepatitis B infection. TAF activation involves multiple steps, and the first step is an ester hydrolysis reaction catalyzed by hydrolases. This study was to determine the contributions of carboxylesterase 1 (CES1) and cathepsin A (CatA) to TAF hydrolysis in the human liver. Our in vitroincubation studies showed that both CatA and CES1 catalyzed TAF hydrolysis in a pH‐dependent manner. At their physiological pH environment, the activity of CatA (pH 5.2) was 700‐1000‐fold higher than that of CES1 (pH 7.2). Given that the hepatic protein expression of CatA was approximately 200‐fold lower than that of CES1, the contribution of CatA to TAF hydrolysis in the human liver was estimated to be much greater than that of CES1, which is contrary to the previous perception that CES1 is the primary hepatic enzyme hydrolyzing TAF. The findings were further supported by a TAF incubation study with the CatA inhibitor telaprevir and the CES1 inhibitor bis‐(p‐nitrophenyl) phosphate. Moreover, an in vitrostudy revealed that the CES1 variant G143E (rs71647871) is a loss‐of‐function variant for CES1‐mediated TAF hydrolysis. In summary, our results suggest that CatA may play a more important role in the hepatic activation of TAF than CES1. Additionally, TAF activation in the liver could be affected by CES1 genetic variation, but the magnitude of impact appears to be limited due to the major contribution of CatA to hepatic TAF activation.
Mapping Spatio‐temporal ADAR Editing Landscapes in Neurodevelopment
Authors: Plonski Noel‐Marie , Meindl Richard , Piontkivska Helen
Published: May 2022

Background

Transcriptome diversity, more specifically RNA editing, plays a fundamental role in neurodevelopment and plasticity. One form of RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADAR), creates variant isoforms, many of which are involved in important pathways including differentiation, proliferation, cell‐migration, vesicular trafficking, and receptor endocytosis and recycling. Immune activation can lead to changes in gene expression of one specific isoform, ADAR1p150, altering RNA editing landscapes causing changes to the structure and/or function of proteins, which, in turn, may contribute to the broad spectrum of symptoms seen in many neurological disorders.

Methods

Using publically available RNA‐seq datasets combined with our computational pipeline, Automated Isoform Diversity Detective (AIDD), including our novel statistical approach using Guttman scaling, we examine global ADAR‐editing patterns in different regions and time points during brain development. In addition, to explore the more subtle nuances of editing changes, nucleotide frequencies at individual genes within the excitome are used to determine differential expression of variant isoform.

Results

ADAR expression and global ADAR editing patterns are significantly different between both brain regions and gestational week. Additionally, ADAR editing landscapes are spatially and temporally regulated, especially prominent in the excitome, a collection of 109 experimentally confirmed ADAR editing sites. A few editing sites are unique to each developing region, while other sites are differentially edited across gestational weeks. Finally, serotonin receptor 2C (5HTR2C), with 5 well‐studied editing sites, is differentially edited early in development in choroid plexus and later in development in the basal ganglia, but not edited in the cerebellum.

Conclusions

Mapping the global RNA editing patterns during neurodevelopment offers insights into mechanisms behind the evolution of higher cognitive function, as well as into molecular pathways underpinning neurological disease. Changes in the excitome variant isoform expression can potentially be used as diagnostic, prognostic and/or therapeutic biomarkers in neurodevelopmental, psychiatric, and degenerative disorders.
The Shape of Silence: A Role for the Nuclear Envelope in Transcriptional Regulation
Authors: Sosa Ponce Maria Laura , Moradi‐Fard Sarah , Cobb Jennifer A. , Zaremberg Vanina
Published: May 2022

The lipid drug edelfosine integrates into the nuclear envelope (NE) of S. cerevisiae (budding yeast) and results in its morphological deformation. As biologists we are guided by a central tenant that form and function are inextricably linked. This is true for the thumb on a hand and a protein produced in a cell. We hypothesized that this will also be true for the organelles inside a eukaryotic cell, wherein changes in nuclear shape would lead to changes in nuclear functions, like alterations in gene expression and protein localization. Using live cell imaging combined with yeast genetics and biochemical approaches, we found that edelfosine treatment led to dispersion of the membrane‐associated SIR complex and triggered processing of the membrane‐sensing transcription factors, Mga2 and Spt23. By a complementary approach of RNA‐seq followed by transcriptomic analysis, we identified that targets of Mga2 as well as subtelomeric regions were upregulated in response to edelfosine, while ribosomal protein targets of Rap1 were downregulated. Our work suggests that disruption of the nuclear membrane is sufficient to trigger changes in membrane‐associated transcription factors and chromatin remodellers. Our data indicate that nuclear membrane integrity is linked to transcriptional regulation. The NE could be a novel target for chemotherapeutics as lipid drugs like edelfosine do not cause DNA damage and would likely not pose mutagenic threats.
TIP60 mediated regulation of ΔNp63α is associated with Cisplatin resistance
Authors: Hira Akshay , Stacy Andrew J. , Craig Michael , Zhang Jin , Kadakia Madhavi
Published: May 2022

About 5.4 million basal and squamous cell skin cancers are diagnosed each year in the US. Cisplatin, a chemotherapeutic drug is often used to treat squamous cell carcinoma (SCC) patients. However, 55% of cancers fail to respond leading to lower response rates and higher rates of disease re‐occurrence. ΔNp63α a member of the p53 transcription factor family is overexpressed and considered oncogenic in non‐melanoma skin cancer where it regulates cell survival and proliferation. TIP60 (Tat‐interacting protein 60kDa), a histone acetyltransferase has been shown to mediate cellular processes such as transcription regulation and the DNA damage response (DDR). We recently reported that TIP60 positively regulates ΔNp63α protein levels in a catalytic‐dependent manner to promote SCC proliferation. Since ΔNp63α is known to transcriptionally regulate several DDR genes and promote resistance to cisplatin, its stabilization by TIP60 may contribute to the failure of platinum‐based drugs in SCC. We hypothesis that TIP60 mediated acetylation of ΔNp63α regulates its transcriptional activity thereby modulates chemoresistance. In this study, we showed that silencing of endogenous TIP60 in multiple SCC cell lines led to decrease in ΔNp63α transcript and protein levels confirming that TIP60 positively regulates ΔNp63α. Increased levels of TIP60 positively correlated with increased ΔNp63α expression and contributes to cisplatin resistance. Further, stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, whereas loss of ΔNp63α and TIP60 sensitized cells to cisplatin. Higher acetylation of ΔNp63α and TIP60 were seen in cisplatin resistant cell line. Taken together, our data suggest that TIP60‐mediated stabilization of ΔNp63α increases cisplatin resistance and has potential implication for SCC cancer treatment and drug design. Additionally, since ΔNp63α confers cisplatin resistance through regulation of genes involved in DNA damage repair, our findings provide critical insights into the mechanism by which genes involved in chemoresistance are regulated and may lead to strategic treatment for resistant SCC tumors and other epithelial cancers with increased efficacy.
Characterization of the DH475 cooperating antibody and its interaction with the HIV‐1 spike
Authors: Finkelstein Maxwell T. , Erdman Molly C. , Fera Daniela
Published: May 2022

HIV evolves very quickly, so approaches to design an effective vaccine that elicits protective antibodies have thus far been unsuccessful. Current HIV vaccine design efforts seek to elicit broadly neutralizing antibodies, unique antibodies that target many viral variants, by first eliciting their precursors through prime‐boost regimens. This requires an understanding of the co‐evolution between viruses and antibodies. Towards this goal, we are analyzing a cooperating antibody, called DH475, which exerted pressure on HIV to evolve in such a way that it became sensitive to the DH270 broadly neutralizing antibody lineage. This study aims to elucidate how DH475 binds to the HIV viral spike and identify how DH475 facilitated the development of DH270 broadly neutralizing antibodies. We obtained a 2.90Å crystal structure of DH475 in complex with Man9 glycan, and used site‐directed mutagenesis coupled with biolayer interferometry (BLI) and protein‐protein docking to characterize how DH475 interacts with Env. These investigations revealed a glycan‐dependent epitope, and docking analyses identified an unorthodox binding mode in which the DH475 framework and constant regions participate in binding. While further confirmation of DH475’s binding mode is required, our findings indicate an overlapping epitope between the DH270 lineage and DH475, consistent with its cooperative neutralization ability.
Discovery of a Fungal Metabolite as a Novel TMEM16a Inhibitor in Human Airway Epithelial Cells
Authors: Pongkorpsakol Pawin , Satianrapapong Wilasinee , Muanprasat Chatchai
Published: May 2022

Transmembrane protein 16a (TMEM16a) is a Ca2+‐activated Cl‐ channel (CaCC) that expresses in various epithelia. Inhibition of TMEM16a has been reported as a potential strategy for attenuating severity and progression of asthma. The main objective of this study is to discover a novel TMEM16a inhibitor and mechanisms of its action. By screening, we found that N9, a fungal metabolite, inhibited UTP‐activated TMEM16a Cl‐ current in basolaterally permeabilized Calu‐3 cell monolayers with an IC50 of ~ 0.8 µM and maximal inhibitory effect being observed at 10 µM. Indeed, N9 had no cytotoxic effect and did not disrupt airway epithelial barrier function, suggesting that inhibitory effect of N9 on TMEM16a was not due to the disturbance of normal airway epithelial function. Electrophysiological analyses indicated that N9 suppressed both ionomycin‐ and thapsigargin‐activated TMEM16a Cl‐ current. In addition, N9 had no effect on elevated intracellular Ca2+ level induced by UTP and thapsigargin, suggesting that N9 reduced TMEM16a Cl‐ current in human airway epithelial cell monolayers in a Ca2+‐independent mechanism. Of particular importance, N9 also significantly suppressed TMEM16a‐mediated Cl‐ current induced by Eact, a direct activator of TMEM16a, indicating that N9 exerts a novel class of TMEM16a inhibitor in human airway epithelial cell monolayers. Hence, this study may lead to identification of a novel class of anti‐asthmatic drug from fungal metabolites.
NLRP3 Inhibition Improves Maternal Blood Pressure, Inflammation, and Vascular Function During Placental Ischemia
Authors: Wang Xi , Travis Olivia K. , Shields Corbin A. , Tardo Geilda A. , Giachelli Chelsea , Nutter Christopher W. , Glenn Hannah L. , Cooper Olive G. , Davis Tatiana , Davis Rashauna , Williams Jan M. , Cornelius Denise C.
Published: May 2022

Preeclampsia (PE) is a pregnancy, hypertensive disorder with end‐organ damage, that develops after 20 weeks of gestation. PE pathophysiology often includes vascular dysfunction and increased inflammation that continues to damage patient health even after their PE resolves. Currently, there are no treatments for PE beyond delivery of the fetal‐placental unit. Previous clinical studies have identified elevated NLRP3 expression in PE placentas and suggest NLRP3 as a potential therapeutic target. In this study, we examined the effects of NLRP3 inhibition on PE pathophysiology in the reduced uterine perfusion pressure (RUPP) rat using MCC950 (20 mg/kg/day) or esomeprazole (3.5 mg/kg/day). We hypothesized that increased NLRP3 in response to placental ischemia impairs anti‐inflammatory IL‐33 signaling to induce T‐helper17 cell (TH17) and cytolytic NK cell (cNK) activation, which is known to mediate oxidative stress and vascular dysfunction causing maternal HTN and intrauterine growth restriction. RUPP rats had significantly higher placental NLRP3 expression, maternal blood pressure, fetal reabsorption rate, vascular resistance, oxidative stress, cNKs and TH17s, and decreased IL‐33 compared to normal pregnant (NP) rats. NLRP3 inhibition, with either treatment, significantly reduced placental NLRP3 expression, maternal blood pressure, fetal reabsorption rates, vascular resistance, oxidative stress, cNK and TH17 populations in RUPP rats. Based on our findings, NLRP3 inhibition reduces PE pathophysiology and esomeprazole may be a potential therapeutic for PE treatment.
Hydrostatic Pressure Controls Angiogenesis through Endothelial YAP1 during Lung Regeneration
Authors: Mammoto Tadanori , Hunyenyiwa Tendai , Kyi Priscilla , Hendee Kathryn , Matus Kienna , Mammoto Akiko
Published: May 2022

Pulmonary artery (PA) pressure increases during lung growth after unilateral pneumonectomy (PNX). The objective of this study is to understand the mechanism by which increases in PA pressure control angiogenesis during lung regeneration. Mechanosensitive transcriptional co‐activator, yes‐associated protein (YAP1), in endothelial cells (ECs) is necessary for angiogenesis during post‐PNX lung growth. We investigate whether increases in PA pressure following PNX controls angiogenesis through YAP1. When hydrostatic pressure is applied to human pulmonary arterial ECs (HPAECs), the expression of YAP1, transcription factor TEAD1, and angiogenic factor receptor Tie2 increases, while these effects are inhibited when HPAECs are treated with YAP1 siRNA or YAP1S94A mutant that fails to bind to TEAD1. Hydrostatic pressure also stimulates DNA synthesis and cell migration in HPAECs, while YAP1 knockdown or YAP1S94A mutant inhibits the effects. Gene enrichment analysis reveals that the levels of extracellular matrix or cell adhesion molecules are altered in post‐PNX mouse lung ECs, which interact with YAP1. These results suggest that increases in PA pressure stimulate angiogenesis through YAP1 during regenerative lung growth.
Functional MYO5B Loss Alters Lipid Metabolic Pathways in Intestinal Progenitor Cells
Authors: Kaji Izumi
Published: May 2022

Background and Aims

: Differentiation of intestinal epithelial cells involves cell division inhibition, cell lineage choice, and brush border elaboration. Myosin Vb (MYO5B) is a motor protein that is critical for cell polarization and membrane protein trafficking in intestinal epithelial cells and its deficiency causes both hyperproliferation and microvillus defects. Recently, we reported that a bioactive phospholipid, lysophosphatidic acid (LPA), can promote cell differentiation and nutrient absorption in tamoxifen‐induced MYO5B knockout mice in vivo and in enteroids. Our RNA‐sequencing data with KEGG pathway and GO enrichment analysis indicated that MYO5B loss disrupts stem cell characteristics, cell lineage differentiation, and energy metabolic pathways. We hypothesize that the alteration of metabolic pathways in progenitor cells causes the disruption of stem cell function.

Methods

: Adult Villin‐CreERT2;Myo5bflox/flox or littermate control (Myo5bflox/flox) mice received a single dose of tamoxifen and the intestinal tissues were analyzed 4 days after the tamoxifen injection. Fresh frozen sections of jejunum were analyzed by imaging mass spectrometry (IMS). Immunostaining for key metabolic enzymes were utilized to support the RNA‐seq and IMS observations.

Results

: Using RNA‐sequencing, MYO5B deficient epithelial cells showed significant decreases in transcripts for enzymes that mediate fatty acid ß‐oxidation compared to control mouse jejunum. Instead, Acsl3, Acat2, and Hmgcs2 were significantly upregulated, proteins that are important for lipogenesis, cholesterol ester synthesis, and ketogenesis, respectively. Consistent with these results, IMS demonstrated that MYO5B deficient tissues accumulated long‐chain fatty acids, phosphatidylinositol, cholesterol derivatives, and nucleotide products in the mucosa (Figure 1). Immunostaining signals of HMGCS2 in control mouse jejunum is limited to the crypt stem cells, whereas MYO5B‐deficient intestine showed expanded HMGCS2 expression in the proliferative cell zone. Interestingly, systemic LPA treatment in MYO5B‐deficient mice further increased the expression of HMGCS2 in both proliferating and differentiated epithelial cells (Figure 2). These observations suggest that MYO5B loss induces a starvation‐like phenotype in proliferative epithelial cells and that LPA treatment increased ketone body production as alternative energy fuels, resulting in the promotion of epithelial cell differentiation.

Conclusion

: MYO5B loss impairs fatty acid oxidation in the intestinal progenitor cells, which likely leads to cell differentiation deficits. Alteration of metabolic pathways might be a therapeutic target for epithelial disfunction seen in diarrheal diseases.
Different modes of Piezo1 Ca2+ signaling in principal and intercalated cells of the collecting duct
Authors: Tomilin Viktor N. , Pyrshev Kyrylo , Stavniichuk Anna , Zaika Oleg , Pochynyuk Oleh
Published: May 2022

It has been recognized that mechanical stress arising from elevated fluid flow and variations in osmolarity is an important regulator of the water‐electrolyte transport in the real tubule. The collecting duct is the terminal segment of the renal tubule, which is commonly exposed to the highest level of mechanical forces. It consists of electrically uncoupled principal and intercalated cells having different morphology and discrete functional roles. Using genetic animal models, we previously demonstrated that mechano‐activated TRPV4 and TRPC3 Ca2+‐permeable channels mediate responses to high flow and hypotonicity, respectively. However, both channels are not considered to be truly mechanosensitive, but rather being activated in response to a primary putative mechanosensor. Piezo1 has been recently identified as a Ca2+‐permeable channel directly activated by the plasma membrane stretch. Piezo1 expression has been found in the renal epithelia, but its role in mechanosensitive Ca2+ signaling is not known. Here, we used freshly isolated split‐opened cortical collecting ducts to show that selective Piezo1 activators, YODA‐1(20uM) and Jedi2 (500uM) acutely increase [Ca2+]i in both principal and intercalated cells. The responses were abolished when extracellular Ca2+ was buffered with EGTA suggesting a direct Ca2+ influx via Piezo1. Interestingly, we detected two disparate calcium responses to Piezo1 activation with the majority (70%) cells showing a fast transient response with a sustained plateau, whereas the remaining (30%) population had much slower gradual calcium increase, to the same plateau level. The subsequent immunofluorescent analysis revealed that the cells with rapid responses to Piezo1 stimulation express AQP2, a selective marker of principal cells. In contrast, AQP2‐negative intercalated cells matched the population of cells with the slow response. Genetic deletion and pharmacological inhibition of TRPV4 and TRPC3 (with GSK2193874 and Pyr10, respectively) did not affect Piezo1‐dependent elevations in [Ca2+]i arguing against a functional link between channel activation with flow‐ and osmo‐sensitive Ca2+ signaling. In summary, our results demonstrate that Piezo1 is functional in both principal and intercalated cells of the collecting duct with a greater expression in the former. Activation of the channel is not directly associated with mechanosensitive Ca2+ signaling via TRPV4 and TRPC3. Future studies are necessary to unravel the significance and physiological roles of different Piezo1 expression in principal and intercalated cells of the collecting duct.