bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic Analysis of Human Breast Milk to Reveal Potential Protein Biomarkers for Breast Cancer
Authors: Whitham Danielle , Aslebagh Roshanak , Channaveerappa Devika , Pentecost Brian , Arcaro Kathleen F. , Darie Costel C.
Published: May 2022

Breast cancer (BC), one of the most common cancers, is a leading cause of death for women in the United States. An estimated 1 in 8 women in the United States will develop BC in their lifetime. Early diagnosis and treatment of BC is crucial, and protein biomarkers for this disease could make this possible. Mass spectrometry (MS)‐based proteomic methods are ideal for the investigation of protein biomarkers. This study employs MS‐based proteomics to study the protein differences in human breast milk from women with BC and matched controls. If significant protein dysregulations are revealed, they could be considered potential future protein biomarkers of BC for diagnosis and treatment. In this study, six human breast milk samples were analyzed by performing a 2D‐SDS‐PAGE and further analyzed via nanoLC‐MS/MS. The human breast milk samples consisted of three comparison pairs of BC vs. control. There were one Coomassie blue‐stained gel (6 total gels) and two replicate silver‐stained gels (12 total gels) for each sample of breast milk. The silver‐stained gels were scanned using a laser densitometer and the images were analyzed using Progenesis Same Spots and Progenesis PG240 software. The spot percentages were measured for each dysregulated spot., An in‐gel trypsin digestion was performed for each spot with statistically significant dysregulation. This was followed by nanoLC‐MS/MS, data processing, database search and statistical analysis. The dysregulated proteins found in this study can be investigated as BC biomarkers for future clinical methods for early diagnosis and treatment of BC.
Biodistribution and Efficacy of an NF‐κB‐Targeted Biologic for Therapy of Ischemic Stroke
Authors: Howell John A. , Perkins Eddie , Burke Stephen , Lopez Mariper , Bidwell Gene L.
Published: May 2022

Strokes are the fifth leading cause of death in the United States, and ~87% of strokes are ischemic, meaning that they are caused by occlusion of a cerebral artery or its branches. This occlusion results in a lack of oxygen reaching the tissue, increases in intracellular calcium and glutamate release, and the activation of inflammatory processes, which can cause additional tissue loss and worsen neurological condition. The nuclear factor kappa B (NF‐κB) cascade is an inflammatory pathway activated by many cytokines and inflammatory signals that are elevated following ischemic stroke, making it a central hub for post‐stroke inflammation. While there are current therapies to treat the occlusion causing an ischemic stroke, they do not treat the inflammation induced injury that occurs. We have designed a novel inhibitor of the NF‐κB cascade attached to the carrier protein elastin‐like polypeptide (ELP) as an adjuvant to current revascularization therapies. ELP is based on the sequence of human elastin, has a long plasma half‐life, is thermally responsive, and has a tunable size. We hypothesize that ELPs can be used to deliver our inhibitory peptide SynB1‐ELP‐p50i to the infarcted region after ischemic stroke, reduce inflammation, and spare brain tissue. To begin, BV2 microglia, C8‐D1A astrocytes, and SH‐SY5Y neurons were treated with rhodamine labeled SynB1‐ELP‐p50i for 24 hours and imaged with confocal microscopy to determine if the protein was able to enter the cells. SynB1‐ELP‐p50i entered the cells and was primarily localized to the cytoplasm. Next, using the middle cerebral artery occlusion (MCAO) model of ischemic stroke in spontaneously hypertensive rats (SHRs), biodistribution and pharmacokinetics were assessed following a bolus administration of 50 mg/kg rhodamine labeled SynB1‐ELP‐p50i, using two routes of administration (intravenous (IV, femoral vein) and intraarterial (IA, carotid catheter)). SynB1‐ELP‐p50i accumulated in the ischemic hemisphere at levels nearly 8 fold higher than in the contralateral hemisphere following MCAO (two‐way ANOVA F(1,12) = 63.08; Sidak’s p < 0.0001). There was no difference in intrabrain deposition between IV and IA routes. To assess the efficacy of SynB1‐ELP‐p50i (50 mg/kg, IV) to reduce infarct size and inflammation following MCAO, the brains were stained, and infarct size was measured using Image J. There was no effect of SynB1‐ELP‐p50i treatment on infarct size compared to saline controls, and the baseline mean arterial pressures were the same between treatment groups. qPCR analysis of downstream targets of the NF‐κB cascade are ongoing to determine if SynB1‐ELP‐p50i treatment affected the production of inflammatory cytokines following MCAO. Future studies will assess effects of SynB1‐ELP‐p50i treatment on behavior and neurological deficits following MCAO in SHRs.
Protein kinase G phosphorylation of Chip protects ovariectomized females from myocardial infarction
Authors: McKoy Desirae M.
Published: May 2022

Proteotoxicity from insufficient clearance of misfolded/damaged proteins exacerbates cardiac functional decline following a myocardial infarction (MI). Estrogen is known to play a crucial role in the survival rate of cardiomyocytes following a MI and is associated with increased protein degradation, however the mechanisms remain to be fully elucidated. Further estrogen has been shown to enhance myocardial protein kinase G (PKG) activity following an insult. In this study, we link this cardioprotective signaling pathway to the carboxyl terminus of Hsc70‐interacting protein (Chip), specifically via phosphorylation at serine 20 (pS20). Chip is a unique effector of proteostasisfunctioning as both a co‐chaperone and ubiquitin ligase. We test the role of Chip S20 with phospho‐mimetic (SE) and phospho‐null (SA) mice compared to WT mice in ovariectomized (OVX) and intact female mice following a MI. We did not observe anydifferences in cardiac function or proteostasis in sham mice regardless of genotype and OVX/intact surgery. Chip SE mice were protected against a MI in both OVX and intact female mice, as exhibited by maintained cardiac function and morphology. Chip WT, even moreso in Chip SA OVX mice, presented with decreased cardiac function, left ventricular chamber dilation and wall thinning, and myocardial fibrosis. Ubiquitinated proteins and protein aggregates were increased in the hearts of Chip SA OVX micewhile Chip SE OVX mice had little accumulation of either, both compared to Chip WT OVX mice. Notably the phospho‐status of Chip S20 did not alter the degradative capacities of the proteasome or lysosome, suggesting the underlying mechanism for enhanced proteostasis is due to increase functionality of the Chip complex. Collectively these dataindicate phosphorylation of Chip S20 is a critical regulator of cardiac proteostasis and is able to afford protection to OVX mice following a MI. These findings have clinical potential as post‐menopausal women are most at risk for deleterious outcomes following a heart attack.
GABAergic and Glycinergic Synaptic Transmission in Respiratory‐Controlling Kolliker‐Fuse Neurons in Female and Male Rett Syndrome Mice
Authors: Whitaker‐Fornek Jessica R. , Levitt Erica S.
Published: May 2022

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by severe motor behavior disturbances including breathing problems. RTT is caused by silencing mutations in the methyl‐CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker‐Fuse (KF) area of RTT mice, there are fewer GABAergic axon terminals and injection of a GABA reuptake antagonist alleviates breathing problems in vivo. However, no one has examined the physiology of GABA or glycine synapses in the KF of RTT mice. In the present work, we tested the hypothesis that GABAergic and glycinergic synapses are deficient in the KF of RTT mice. We performed whole‐cell voltage clamp recordings from KF neurons contained in brain slices of MeCP2‐deficient RTT mice and wild type littermates. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the KF neurons of male RTT mice was reduced compared to wild type (WT) littermates. In contrast, female RTT mice showed no change in sIPSC frequency in the KF compared to control, despite having breathing abnormalities. The amplitude of sIPSCs in the KF of female mice were smaller compared to WT littermates, whereas male RTT mice showed no change in amplitude compared to WT littermates. These results spotlight the importance of studying respiratory controlling KF neurons in both male and female RTT mice, and suggest that reductions in IPSC frequency and amplitude are not sufficient to explain the breathing irregularities observed in RTT in females and males, respectively.
Aerobic Fitness as a Predictor of Conduit, Resistance and, Microvascular Arterial Function
Authors: Hibner Brooks A. , Lefferts Elizabeth C. , Lima Natalia S. , Sherman Sara R. , DeJonge Sydney R. , Baynard Tracy , Fernhall Bo
Published: May 2022

Introduction

Vascular dysfunction increases cardiovascular disease (CVD) risk, whereas aerobic fitness (VO2peak) improves arterial function and decreases CVD risk. However, it is unclear if VO2peak has the same degree of importance across each segment of the arterial tree (e.g., conduit, resistance, and microvasculature). This is relevant given that research has not assessed these three segments in the same cohort.

Purpose

To examine the relationship of VO2peak and conduit, resistance, and microvasculature arterial function.

Methods

Graded exercise testing assessed VO2peak in 33 males (41±19 years, 25.6±2.6 kg/m‐2, 41.1±13.6 ml/kg‐1/min‐1) and 28 females (30±12 years, 23.3±4.3 kg/m‐2, 33.6±8.5 ml/kg‐1/min‐1). Concurrent brachial artery flow‐mediated dilation (FMD) and forearm near‐infrared spectroscopy (NIRS) examined conduit, resistance, and microvascular arterial function. FMD measured with ultrasonography to continuously record arterial diameters and Doppler velocities for 1 min baseline, 5 min forearm occlusion (250 mmHg), and 3 min recovery. The maximum percent change in diameter compared with baseline defined FMD (%) and was used to evaluate conduit arterial function. Reactive hyperemia (RH) of forearm resistance arteries was calculated as peak brachial blood flow following cuff release. Forearm tissue saturation index using NIRS during FMD was used to evaluate microvasculature function (reperfusion slope and reperfusion magnitude). Multivariate regression analysis was performed using VO2peak, age, sex, and BMI as independent variables.

Results

Age was the only predictor of FMD (β ‐0.48 [‐0.02, ‐0.01], p=0.001), sex was the only predictor of RH, females exhibited lower RH, (β ‐0.66 [‐0.30, ‐0.15], p<0.001), and VO2peak only predicted reperfusion slope (β 0.56 [1.46, 4.52], p< 0.001) and reperfusion magnitude (β 0.36 [3.15, 41.66], p=0.02).

Conclusion

In our cohort, aerobic fitness was associated with microvascular function when age, BMI, and sex were included in the model. Only age was associated with conduit artery function, and only sex was associated with resistance arterial function. Each segment of the arterial tree was affected differently by VO2peak, age, and sex. These data show the importance of including several potentially important variables when investigating the association between VO2peak and arterial function.
Spatial Whole Transcriptome Profiling of the Tumor Microenvironment in Prostate Carcinomas
Authors: Walker Dan , Giangarra Valeria , Anaparthy Naishitha , Williams Stephen , Sheldon Jyoti , Turkekul Mesruh , Mielinis Paulius , Gallant Caroline , Chell James , Taylor Sarah
Published: May 2022

Background

Despite years of studies and effort, the best strategies for treating prostate cancer and minimizing the complications of treatment remain unanswered questions. This gap in knowledge is partially due to the inability to dissect the complex heterogeneous tumor microenvironment (TME) and immune compartment. Spatially resolved molecular profiling of tumor sections will enhance our understanding of these complexities; However, it has been particularly challenging to do spatial molecular profiling in formalin‐fixed paraffin‐embedded (FFPE) tissues due to RNA degradation associated with this tissue‐embedding method, which is routinely used in oncology workflows.

The 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue overcomes these limitations, enabling spatial gene expression analysis of FFPE tissues combined with classical histology staining techniques such as Hematoxylin & Eosin (H&E) staining and immunofluorescence.

Methods

We used the 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue to analyze and resolve tumorigenic profiles in sections of normal and adenocarcinoma prostate samples. This assay incorporates ~5,000 molecularly barcoded, spatially encoded capture probes in spots over which the tissue is placed, imaged, and permeabilized. Imaging and sequencing data are processed together, resulting in a spatially resolved transcriptional readout.

Results

We profiled the whole transcriptome in normal, invasive adenocarcinoma, and acinar cell carcinoma FFPE human prostate tissues.

Unsupervised clustering of the whole transcriptome data from normal, invasive adenocarcinoma, and acinar cell prostate carcinoma FFPE sections enabled the identification of 2 different regions, which had a well defined spatial distribution within the tissues. Well known prostate gland and prostate‐cancer markers were over‐expressed in the corresponding healthy and cancerous portions of the tissue, validating the performance of this method. We found that, while in healthy tissues basal cells and luminal cells are spatially organized, this pattern is lost in tumor samples, where luminal cells are greatly expanded in the invasive carcinoma region and do not colocalize with basal cells. Moreover, T lymphocytes are dispersed throughout the whole tissue section in the adenocarcinoma, while plasma B cells are located in the peritumoral region which could impact prognosis.

Conclusions

Spatial whole transcriptome analysis opens new opportunities for better understanding the TME which can not only help discover novel predictive tumor biomarkers, but also enable identifying cell type and tumor region specific drug targets.
Targeting 5‐HT1A Receptors to Correct Perineuronal Net and Social Behavioral Deficits in Fmr1 Knockout Mice, a Model of Fragile X Syndrome
Authors: Armstrong Jessica L. , Canal Clinton E.
Published: May 2022

Fragile X syndrome (FXS), caused by FMR1gene silencing and subsequent loss of the RNA‐binding protein FMRP, is the leading monogenic cause of intellectual disability and autism spectrum disorder, characterized by social deficits and repetitive behaviors. Currently, there are no approved medications for FXS. MMP‐9 is an extracellular matrix regulating enzyme and a significant target of FMRP—the loss of FMRP in FXS leads to overexpression of MMP‐9. MMP‐9 degrades perineuronal nets (PNNs) that stabilize glutamatergic and GABAergic neurotransmission, and imbalance in these neurotransmitter systems is a hallmark of FXS, suggesting correcting MMP‐9 overactivity may restore neuronal homeostasis in FXS by normalizing PNNs. For example, we show significantly decreased PNN density in hippocampal CA2 in juvenile Fmr1 knockout (KO) mice, a neural system critical for social memory, compared to wild‐type (WT) mice (P=0.0043, N=8 and 7, respectively). In parallel with PNN deficits, we show that juvenile Fmr1KO mice have social memory impairments compared to WT mice. Juvenile Fmr1 KO mice have no preference for interacting with novel mice than familiar mice (number of interactions, P=0.54; amount of time interacting, P=0.27; N=10). In contrast, WT mice interact more frequently and spend more time with novel mice (P=0.014 and P=0.028, respectively; N=11). Modulating distinct serotonin receptors (5‐HTRs) can regulate MMP‐9 expression, neuroanatomical plasticity, social behaviors, and memory. Previously we showed that FPT, a novel medication candidate with potent 5‐HT1AR agonist activity, increases social interactions in adult Fmr1KO and WT mice (Armstrong et al., 2020 https://doi.org/10.1021/acsptsci.9b00101). However, we did not investigate FPT’s effects on social behavior or social memory in juvenile mice—focusing on critical neurodevelopmental periods—nor have we explored mechanisms underlying FPT’s therapeutic‐like effects. We are now testing the hypothesis that FPT corrects social memory deficits in juvenile Fmr1 KO mice and works, in part, by decreasing MMP‐9 levels and restoring PNN levels in hippocampal CA2 via a 5‐HT1AR mechanism. Results from these studies will provide critical mechanistic information regarding the preclinical neurotherapeutic effects of FPT in FXS.
Assessing the Antinociceptive Effects and Tolerance Development of Decursinol in Nociceptive, Inflammatory, and Neuropathic Pain
Authors: Crawford LaTaijah , Henderson‐Redmond Angela , Kim Sangyub , Karelia Deepkamal , Sepulveda Diana , Lu Junxuan , Morgan Daniel
Published: May 2022

Chemotherapy‐induced neuropathic pain is a prevalent side effect of treatment with anti‐neoplastic agents. This debilitating neuropathy is characterized by excessive pain resulting in mechanical and sensory hypersensitivity. There is a critical clinical need to develop non‐opioid alternatives to manage chronic pain. A potential alternative for the management of pain is decursinol, a pyranocoumarin compound isolated from the root of the Angelica gigas Nakai (AGN) plant. Pharmacokinetic studies find that clinically, AGN is first metabolized into decursin (D) and decursinol angelate (DA) which are subsequently metabolized into decursinol (DOH). D and DA possess anti‐inflammatory, neuroprotective, antinociceptive, and antioxidant modulatory properties. Studies have reported acute analgesic properties of DOH, but tolerance development along with its impact on chronic pain remains unknown. Therefore, the goal of this study was to assess the antinociceptive effects and potential tolerance to once‐daily injections of DOH (50 mg/kg) in models of thermal (hot plate and tail‐flick), inflammatory (formalin test), and chronic (cisplatin‐induced neuropathic; CINP) pain. We hypothesized that DOH has prolonged antinociceptive and anti‐inflammatory effects. Antinociception and tolerance were assessed in male mice treated once‐daily with either vehicle, DOH (50 mg/kg), or morphine (10 mg/kg; formalin only) intraperitoneally (IP) 30 minutes prior to testing for 14 consecutive days. In the formalin assay, following pretreatment, mice received an intraplanar injection of 2.5% formalin into the right hind paw and nocifensive behaviors were recorded and scored. CINP was induced following four weeks of once‐weekly IP injections of cisplatin (5 mg/kg). Following pretreatment, mice with CINP were assessed for mechanical allodynia via an electro‐von‐Frey anesthesiometer. Daily treatment with 50 mg/kg DOH induced prolonged antinociceptive and anti‐inflammatory responses in all nociceptive tests.

Evidence of tolerance to DOH in acute thermal nociceptive tests occurred around day 10 for both hot plate and tail‐flick. Acute administration of DOH eliminated inflammatory pain to an undetectable level in both the acute and inflammatory phases of the formalin test. Further, there is evidence of tolerance by day 14 in the acute (but not the inflammatory) phase of the formalin test. Likewise, tolerance was slower to develop to the antinociceptive effects of DOH compared to morphine in the formalin assay. In mice with CINP, DOH (50 mg/kg) caused a full reversal of mechanical allodynia with evidence of partial tolerance by day 3 with complete tolerance not occurring until day 14. These data indicate that DOH may serve as a potential non‐opioid alternative for the management of pain. Future directions for this study include elucidating the mechanism of action for the antinociceptive effects of DOH.
Role of β‐Arrestin2 as a Regulator of Fluid Homeostasis and Blood Pressure
Authors: Mathieu Natalia M. , Sigmund Curt D. , Nakagawa Pablo , Grobe Justin L.
Published: May 2022

Low‐renin hypertension is a common subset of hypertensive patients, which is associated with salt sensitivity. Deoxycorticosterone (DOCA)‐salt hypertension, a prototypical model of low‐renin hypertension, requires activation of the angiotensin type‐1 receptor (AT1R) in specific brain areas. The canonical G‐protein‐mediated signaling of the AT1R is known to induce pressor responses while the non‐canonical β‐Arrestin mediated signaling is thought to counterbalance the detrimental effects of canonical signaling. We hypothesized that β‐Arrestin activation contributes to fluid homeostasis and blood pressure (BP) regulation in DOCA‐salt hypertension. Global β‐Arrestin1 (Arrb1) and β‐Arrestin2 (Arrb2) knockout (KO) mice were employed to evaluate drinking behavior and BP. Age‐ and sex‐matched C57BL/6J mice served as controls. Mice were subjected to the two‐bottle choice paradigm, in which the animals were presented with two bottles, one containing water and one containing varying concentrations of NaCl (0.15, 0.30, and 0.45 mol/L). At baseline, Arrb2‐KO mice exhibited a significant increase in saline intake when presented with 0.15M and 0.30M but not 0.45M (WT0.15M=2.4±0.8 and WT0.30M 1.2±0.4 vs Arrb2‐KO0.15M=4±1.7 and Arrb2‐KO0.30M=2.3±1.1 mL/day; p0.15M=0.23 and p0.30M<0.05; n=19 and 24, respectively). This resulted in a saline preference, which means mice preferred saline over water by more than 50% by volume. Under DOCA, Arrb2‐KO, but not Arrb1‐KO, exhibited a significant increase in saline intake when presented with 0.15M and 0.30M, but not 0.45M saline (WT0.15M=10.9±3.6 and WT0.30M 3.8±2 vs Arrb2‐KO0.15M=15.2±6.3 and Arrb2‐KO0.30M=6±2 mL/day; p<0.05; n=19 and 24, respectively) when compared with wildtype. In addition, Arrb2‐KO mice showed a significant increase in fluid intake when presented to 0.15M, 0.30M, and 0.45M saline. Telemetric BP was measured under three conditions: 1) baseline without saline in drinking water 2) baseline with a choice of saline and water, and 3) administration with DOCA with a choice of saline and water. Arrb2‐KO did not exhibit significant differences in BP in absence of DOCA infusion when given water alone or a choice for saline. However, Arrb2‐KO exhibited an increase in the pressor response to DOCA‐salt (WT=152.2±11.9 vs Arrb2‐KO=163.7±4.2 mmHg; p=0.18; n=6). Surprisingly, no alterations in water intake, saline intake, total fluid intake, saline preference, or BP was observed in Arrb1 KO at any given saline concentration under either baseline or DOCA condition. These findings suggest that in low renin hypertension, Arrb2, but not Arrb1, might counterbalance the detrimental canonical signaling of pathological G protein‐coupled receptors including AT1R.
MicroRNAs Regulate Hypothalamic AT2R Expression in Hypercaloric Diet‐Induced Epigenetic Programming of Male Mice Offspring
Authors: Elgazzaz Mona , Berdasco Clara , Lazartigues Eric
Published: May 2022

One in ten adults are living with diabetes worldwide and it is expected to be one in eight adults by the year 2045. Maternal epigenetic programming is one of the determinants of disease susceptibility in offspring. The renin‐angiotensin system contributes to metabolic homeostasis through the balance between Angiotensin II (Ang‐II) binding to Angiotensin II Type 1 receptor (AT1R) and Angiotensin II Type 2 receptor (AT2R). We have shown that maternal hypercaloric (HCD) diet leads to a pre‐diabetic state exclusively in adult male offspring. Additionally, we showed a paradoxical reversal of hyperglycemia in programmed males challenged with Ang‐II. Here, we hypothesize that this hypoglycemic response in programmed males is due to a shift in Ang‐II binding from AT1R towards the protective AT2R. To test this hypothesis, C57BL6/J dams were fed HCD or regular diet (RD) for 1 month and mated with RD‐fed males. After weaning, offspring were maintained on RD for 3 months resulting in two groups: HCD‐RD (programmed) and RD‐RD (control). Half of each group was implanted with a subcutaneous Ang‐II osmotic pump (200 ng/kg/min/2 weeks). Another osmotic pump filled with AT2R blocker, PD123319 (10 mg/kg/day) was implanted in a subgroup of Ang‐II programmed mice resulting in 5 groups: HCD‐RD, RD‐RD, HCD‐RD+Ang‐II, RD‐RD+Ang‐II and HCD‐RD+Ang‐II+PD123319 (n=7‐10/group). A glucose tolerance test was performed before and after drugs infusion. Following euthanasia, half of each hypothalamus was harvested for protein quantification using simple western while the other halves were processed for microRNA (miRNA) sequencing. Results show that HCD‐RD+Ang‐II+PD123319 had elevated glucose levels compared to HCD‐RD+Ang‐II mice at 15 min (355.7 ± 32 vs. 279.8 ± 19.66 mg/dL, p=0.04), 30 min (405 ± 18.54 vs. 263.5 ± 22.68mg/dL, p=0.0013) and 60 min (298.3 ± 19.75 vs.189.8 ± 21.35 mg/dL, p=0.0036). On the other hand, there was no difference in the glycemic levels of HCD‐RD+Ang‐II+PD123319 compared to RD‐RD+Ang‐II groups at 15 min (355.7 ± 32.97 vs. 349.0 ± 32.06 mg/dL, p=0.8), 30 min (405.0 ± 18.54 vs. 405.8 ± 44.28 mg/dL, p=0.9) and 60 min (298.3 ± 19.75 vs. 230.0 ± 35.52 mg/dL, p=0.1). AT2R protein expression was higher in the HCD‐RD compared to RD‐RD (p=0.04) and in HCD‐RD+Ang‐II compared to RD‐RD+Ang‐II (p=0.03). For miRNA sequencing, miRNA 124‐3p, targeting AT2R was differentially expressed in the HCD‐RD compared to RD‐RD (FC=1.4, p=0.04) as well as HCD‐RD compared to HCD‐RD+Ang‐II (FC=0.74, p=0.0001). Whereas, no change in the miRNA expression was depicted in the non‐programmed groups; RD‐RD vs RD‐RD+Ang‐II (FC=0.97, p=0.85). In conclusion, maternal hypercaloric diet epigenetic programming takes place probably through regulating the gene expression of AT2R via changing miRNA ‐124 levels that works on the post‐transcriptional modification level.
The Role of PER‐1, ET‐1 Axis in Regulating Endothelial Cell Function
Authors: Jakkidi Nishka R. , Gomes Joshua , Sharma Ravindra K. , Li Shiyu , Gumz Michelle L. , Mohandas Rajesh
Published: May 2022

Objective

Results from major epidemiological studies give evidence to the essential role that dietary salt plays in the development of hypertension. Contrastingly, the mechanisms that mediate individual susceptibility to salt‐sensitive hypertension are unknown. Blood pressure (BP) and sodium reabsorption by the kidneys are regulated by the circadian clock. Consistent with these observations, when mice with global knockout (KO) of the circadian protein period 1 (PER1) are exposed to a high salt diet, they demonstrate increased endothelin‐1 (ET‐1) levels, impaired natriuresis, and hypertension. However, the cumulative sodium balance of PER1 KO mice is not different from control mice. Thus, we hypothesize that PER1 might mediate endothelial cell dysfunction and susceptibility to salt sensitive hypertension.

Materials and Methods

Human Umbilical Vein Endothelial Cells (HUVEC) were transfected with siRNA targeting PER1 or non‐targeting siRNA controls. Endothelial cell function was assessed by measurement of nitric oxide bioavailability, proliferation, and migration. Nitric Oxide (NO) was quantified by DAF fluorescence, proliferation by EZQuant™, and migration by measuring velocity and distance travelled by single cells using time lapse imaging.

Results

Nitric oxide bioavailability was increased with PER1 knockdown (Mean fluorescence 14433 ± 1344 vs. 7423 ± 472, p<0.005). Migration velocity and distance were decreased with PER1 siRNA compared to non‐target controls, though not statistically significant (Mean distance 118.9 uM ± 11.39 vs. 138 uM ± 10.88, p=0.2) and (Mean velocity 42.3 uM/sec ± 3.98 vs. 46 uM/sec ± 3.6, p=0.14).

There were no differences in proliferation at 24, 48, or 72 hours (Fold change in cell numbers of PER1 siRNA treated cells compared to non‐target controls were 0.87 ± .05 at 24 hours, 0.87 ± .09 at 48 hours, and 0.90 ± .06 at 72 hours, p=NS).

Conclusions

Our results suggest that knock down of PER1 increases NO bioavailability and decreases migration of endothelial cells. ET‐1 can increase NO bioavailability via ETB receptors. Experiments are ongoing to delineate the role of ET‐1/ETA and ET‐B in mediating the effect of PER1 on endothelial cell function.
A mechanism of ACK1 activation in cancer via C‐terminal UBA domain truncation
Authors: Balasooriya Eranga , Eastmond Reilly , Madusanka Deshan , Owen Jacob , Gashler Jack , Lopez‐Palacios Tania , Andersen Joshua
Published: May 2022

We recently developed a machine learning (ML) based program to predict cancer hotspots. Here, we applied the ML program to 32 non‐receptor tyrosine kinases (NRTKs) and identified 36 potential cancer driver mutations, with high probability mutations in 10 genes, including ABL1, ABL2, JAK1, JAK3, and ACK1. Interestingly among all the NRTKs, ACK1 is the only kinase that, when altered, shows a significant drop in overall survival, supporting the idea that ACK1 is an oncogenic tyrosine kinase. ACK1 is a member of the poorly understood ACK family of NRTKs that also includes TNK1. Although ACK1 is an established oncogene and high‐interest therapeutic target, the exact mechanism of ACK1 regulation is largely unknown and there is still no ACK1 inhibitor in clinical use. The ACK kinase family has a unique domain arrangement with, most notably, a predicted ubiquitin association (UBA) domain at its C‐terminus. The presence of a functional UBA domain on a kinase is unique to the ACK family, but the role of the UBA domain on ACK1 is unknown. Interestingly, the ML program identified the ACK1 UBA‐truncating mutation p633fs* as a cancer hotspot. Our preliminary data suggest that the ACK1 UBA domain helps activate full‐length ACK1 through induced proximity, but also acts as a mechanism of negative feedback by tethering ACK1 to ubiquitinated cargo that is ultimately degraded. Indeed, our preliminary data suggest that truncation of the ACK1 UBA stabilizes ACK1 protein levels, which results in spontaneous ACK1 oligomerization and activation. Thus, our data provide a model to explain how human mutations in ACK1 convert the kinase into an oncogenic driver.
Novel Regulation of Nitric Oxide Synthase 3 (NOS3) Isoform by Acetylation at Lysine 610
Authors: Dunaway Luke S. , Oenick Jared M. , Liu Xiaofen , Hyndman Kelly A. , Pollock Jennifer S.
Published: May 2022

NOS3‐mediated NO regulates endothelial and kidney epithelial function. Lysine acetylation is a dynamic post‐translational modification influencing cell signaling. Our lab and others showed NOS3 is regulated by multiple deacetylases. We therefore sought to uncover novel lysine acetylation sites of NOS3 and investigated lysines in the NOS3 reductase domain, K607, K610, K727, and K731. We hypothesized that deacetylation of one or more of these lysines on NOS3 would impact NO production. In order to test this hypothesis, we created NOS3 constructs containing lysine to arginine mutations at these sites mimicking deacetylation. These plasmids were transfected into COS7 cells and expression was confirmed with Western blot. NO production was assessed by measuring nitrite accumulation by HPLC in the media over 48 hours. Mutation of all four lysines or lysines K607 and K610 dramatically increased NO production whereas K727 and K731 did not change NO production compared to wildtype (pmol NO2/mg protein, WT: 2271 ± 729.3; K607/610/727/731R: 15176 ± 2759, p=0.046 vs WT; K607/610R: 9431 ± 1772, p=0.0019 vs WT; K727/731R: 3523 ± 805.8, p=0.918). We further investigated single mutation of K607 and K610. We found K610R (10564 ± 1883 pmol NO2/mg protein, p=0.002 vs WT), but not K607R (1589 ± 412.0 pmol NO2/mg protein, p>0.99) to have increased NO production compared to WT (1547 ± 180.8 pmol NO2/mg protein). We then hypothesized that acetylation of K610 on NOS3 would decrease NO production compared to deacetylation of K610. We mutated K610 to glutamine mimicking acetylation of this lysine. Contrary to what we hypothesized, we found K610Q to produce greater NO than both WT and K610R (pmol NO2/mg protein, WT: 2196 ± 606.1, K610Q: 30212 ± 880.2, p<0.0001 WT vs K610Q; K610R:13389 ± 1086, p<0.0001 K610R vs K610Q). Previous reports show that amino acids 605‐612 coordinate regulatory phosphorylation sites of NOS3. We therefore investigated NOS3 K610R and K610Q relative to WT at key phosphorylation regulatory sites by western blot. We found K610R and K610Q to have reduced phosphorylation at T495 (RDU; WT: 1.00 ± 0.252; K610R: 0.820 ± 0.251, p=0.0257; K610Q: 0.711 ± 0.205, p=0.0046), increased phosphorylation at S1177 (WT: 1.00 ± 0.125; K610R: 1.58 ± 0.2921, p=0.038; K610Q: 1.93 ± 0.358, p=0.0082), and no change in phosphorylation at S633 (WT: 1.00 ± 0.054; K610R: 0.885 ± 0.0536, p=0.126; K610Q: 0.955 ± 0.0779, p=0.319). Additionally, K610R but not K610Q was found to have increased S615 phosphorylation (WT: 1.00 ± 0.112; K610R: 2.01 ± 0.259, p=0.0003; K610Q: 1.07 ± 0.112, p=0.682). These data suggest acetylation of K610 would significantly increase NO production and regulate NOS3 phosphorylation sites in endothelial cells and/or kidney epithelial cells. We propose that acetylation/deacetylation of K610 would have dramatic consequences for blood pressure regulation as well as water and electrolyte homeostasis.
Proteome profiling of a S‐Nitrosoglutathione reductase (GSNOR) null mutant reveals that aldo‐keto reductases form a new class of enzymes involved in nitric oxide homeostasis
Authors: Treffon Patrick , Rossi Jacopo , Gabellini Giuseppe , Trost Paolo , Zaffagnini Mirko , Vierling Elizabeth
Published: May 2022

Nitric oxide (NO) is a short‐lived gas that acts as a signaling molecule in all higher organisms, including plants. Despite the involvement of NO in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects is lacking. NO and its derivatives impact these physiological processes through reversible S‐nitrosation of critical protein cysteines. In cells, regulation of NO‐levels is predominantly achieved by reaction of reactive nitrogen species (RNS) with glutathione (GSH), thereby forming S‐nitrosoglutathione (GSNO), a principal NO reservoir. Mutation of Arabidopsis thaliana S‐nitrosoglutathione reductase (GSNOR) leads to higher intracellular concentrations of S‐nitrosothiols, confirming that the GSNOR reduction of GSNO is a major route of GSNO catabolism in plants and other eukaryotes1. We demonstrate in Arabidopsisthat absence of GSNOR results in differential regulation of proteins involved in chlorophyll metabolism, the general stress response and photosynthesis2. In addition, our proteomic analysis identified a significant increase in proteins that belong to the aldo‐keto reductase (AKR) protein superfamily, AKR4C8 and 9. Since specific AKRs have been linked to NO metabolism in mammals, we expressed and purified ArabidopsisAKR4C8 and 9 and close homologues AKR4C10 and 11 and determined that they have NADPH‐dependent activity in GSNO and S‐nitroso‐coenzyme A (SNO‐CoA) reduction. Plants lacking GSNOR also show increased activity of NADPH‐dependent GSNO reduction, consistent with increased AKR activity2. To address the in vivo role of these AKRs, CRISPR/Cas9‐mediated mutant lines will be generated, and their capacity to metabolize RNS assessed. In addition, by subjecting AKR mutant plants to different abiotic and nitrosative stresses, results will uncover the importance of AKR proteins in NO homeostasis during both optimal growth and stress conditions. Taken together, these data define a new, NADPH‐dependent component of NO metabolism that may be integrated with NADH‐dependent GSNOR activity to control NO homeostasis in plants and other organisms.

1. Lee, U., Wie, C., Fernandez, B. O., Feelisch, M. & Vierling, E. Modulation of nitrosative stress by S‐nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis. Plant Cell 20, 786–802 (2008).

2. Treffon, P., Rossi, J., Gabellini, G., Trost, P., Zaffagnini, M., Vierling, E. Quantitative proteome profiling of a S‐nitrosoglutathione reductase (GSNOR) null mutant reveals a new class of enzymes involved in nitric oxide homeostasis in plants. Accepted. Front. Plant Sci. (2021).
A Method for Automated Pharmacophore Model 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. Ligand‐based pharmacophore models can be constructed to identify structural commonalities between known bioactive ligands for targets including GPCR. However, structure‐based pharmacophores (which only require a resolved or predictive structure for a protein) have gained more attention as the number of publicly available, high‐resolution GPCR structures have increased (113 unique GPCR represented as of November 24, 2021). Thus, the goal of this study was to develop a method of structure‐based pharmacophore model generation applicable to ligand discovery for GPCR with few known ligands. Pharmacophore models have been generated using the active sites of 8 class A GPCR crystal structures via automated feature annotation of 5 randomly selected functional group fragments in an attempt to sample diverse combinations of pharmacophore features to aid in virtual screening efforts. Each of the 5000 generated pharmacophores was then used to search a database containing active and decoy/inactive compounds for 30 class A GPCR and scored using enrichment factor and goodness‐of‐hit metrics to assess performance. As proof‐of‐principle for this method, pharmacophore models were also generated within two dopamine receptor 2 (D2) crystal structures to elucidate novel ligands possessing activity for this receptor. Application of this method to the set of 8 class A GPCR generated pharmacophore models possessing the theoretical maximum enrichment factor value in both resolved structures (8 of 8 cases) and predictive models (7 of 8 cases), implying that generated pharmacophore models will prove useful in the context of virtual screening. Furthermore, application of this protocol to the D2 receptor resulted in the identification of novel candidate ligands whose activity has been confirmed with two in vitroactivity assays.
Traumatic brain injury in a Drosophila upregulates nitric oxide synthase associated with increased acute behavioral deficits and decreased survival time
Authors: Mackey Tracy , Jones T. B. , Currier Thomas Theresa , Juba Amber N. , Buhlman Lori M.
Published: May 2022

Traumatic brain injury (TBI) is as an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain nitric oxide (NO), where NO activity is implicated in secondary pathology leading to behavioral deficits. In mammals, TBI‐induced NO production activates immune responses and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. TBI in Drosophilahave clearly demonstrated key features of mammalian TBI including temporary incapacitation, disorientation, and activation of innate immunity (inflammation) and autophagy responses observed immediately after injury and related to persisting deficits. We propose to evaluate nitric oxide synthase expression in the absence of vascular disruption and the adaptive immune system to begin creating a model that minimizes extraneous variables to study the longitudinal influence of NO signaling on secondary pathology leading to behavioral deficits where highly targeted interventions can be evaluated.

We optimized a modified high impact trauma (HIT) device on NOS‐GAL4>UAS2xEGFP flies (Bloomington Stock Center, Bloomington, IN) where upregulation of Drosophila nitric oxide synthase (dNOS) expression is associated with increased GFP levels. Ten‐day old flies received TBI from the HIT device at multiple angles (30, 45, 60, 75 degrees) followed by assessment of acute neurological behavior, locomotor activity, brain dNOS levels, twenty‐four‐hour mortality, and survival. Data indicate an increase in brain GFP expression, where robust reproducible levels of dNOS could be recorded after a 75‐degree HIT associated with increased incident of temporary incapacitation, disorientation, changes in acute climbing activity, increased 24‐hour mortality, and decreased survival. Results also indicate an effect on dNOS expression as a function of HIT angle and sex. Ongoing studies quantify levels of TBI‐induced NO activity in the brains. Future studies will evaluate different forms of brain pathology and more extensive behavioral deficits where genetic and pharmacological manipulation can be used to tease out direct effects of NO signaling. Due to conserved signaling cascades between flies and mammals, improved outcomes from modulating NO activity can translate to improved targeted therapies to be tested in mammals.
Clinical and embryological relevance of a unique thyroidea ima artery variation providing collateral circulation to the mediastinum
Authors: Zachwieja Alexandra J. , Benedict Taylor , Ach Therese , Michaels Robin
Published: May 2022

The thyroidea ima artery (TIA) is a highly variable arterial deviation of the blood supply to the thyroid gland with critical implications for surgical neck procedures such as tracheostomy. Though relatively common in the population at large (~3‐10%), most TIA variations are related to the initiation of the artery and whether it emerges from the common sites of the brachiocephalic trunk, aortic arch, right common carotid artery, or another more unique vessel as opposed to its dispersion pattern. Generally, TIA variants supply the thyroid gland, often co‐occurring with absent inferior or superior thyroid arteries. Here, we report on a unique case of a four‐pronged variation of the TIA discovered during an anatomy lab of first year medical students. This variant initiates from the brachiocephalic trunk (diameter 5.3mm, recorded averages 3‐5mm) and has three branches terminating in the thyroid and a fourth branch traveling into the thorax and providing accessory circulation in the mediastinum. Specifically, small arterial branches off of the inferior TIA branch supply the pericardium and surrounding fat deep to the sternum, with anastomoses to the left pericardiophrenic artery. Following the large size of the TIA itself, the thyroid gland of this individual is also markedly increased in size (A‐P diameter 21mm, lateral width 58mm, S‐I diameter 45mm) Potential embryological underpinnings of this variation include overactive HIF‐1α vascular remodeling. The size and spread of this TIA variation provide further support for imaging as a requirement before surgical neck procedures to prevent catastrophic bleeding in the event of a TIA variant.
Investigating the Effects of Vaping and Nicotine’s Block of Kir2.1 on Humerus and Digital Development in Embryonic Mice
Authors: Moehn Kayla , Ozekin Yunus , Bates Emily
Published: May 2022

Kir2.1 is an ion channel that is important for skeletal development. Nicotine, a common ingredient in electronic cigarettes, blocks Kir2.1. However, little research has been done to investigate how vaping nicotine during pregnancy affects embryonic development. We examined how vaping nicotine during pregnancy would affect embryonic skeletal development in mice. Mice pregnant with embryos of three genotypes—wild‐type, Kir2.1+/KO, or Kir2.1KO/KO—received either no vaping treatment, a 2.4% freebase nicotine vaping treatment, or a 3.6% nicotine salt vaping treatment throughout pregnancy. The embryos were dissected at E18.5, and their skeletons were extracted and stained for imaging. The lengths of the humerus bones and number of digital bones were calculated to quantify differences in bone development between groups. We found significant differences in humerus length between the wild‐type no vape and wild‐type 2.4% freebase nicotine groups, as well as the Kir2.1+/KO no vape and Kir2.1+/KO 2.4% freebase nicotine groups. However, no significant differences were found between wild‐type no vape and wild‐type 3.6% nicotine salt groups, as well as Kir2.1+/KO no vape and Kir2.1+/KO 3.6% nicotine salt groups. We suspect that nicotine salts are metabolized faster than freebase nicotine and thus inhibit Kir2.1’s function for a shorter amount of time, but further research needs to be conducted to test this hypothesis. We conclude that fetal exposure to vaped free base nicotine impacts the development of long bones and reduces long bone length.
A 3D Anatomical and Molecular Map of Cardiac Vagal Motoneurons
Authors: Hornung Eden , Robbins Shaina , Achanta Sirisha , Srivastava Ankita , Moss Alison , Schwaber James , Vadigepalli Rajanikanth
Published: May 2022

Recent efforts through the NIH SPARC program have opened new opportunities for harnessing vagal activity to promote cardioprotection. However, cardiac‐projecting vagal motoneurons of the Dorsal Motor Nucleus of the Vagus (DMV) are relatively understudied, even as recent studies show that DMV activity is required for cardioprotection induced by physiological interventions. By combining principles of neuroscience and single cell transcriptomics, we begin to explore the transcriptomic phenotypes of cardiac‐projecting DMV neurons. Neuromodulatory co‐transmission is an emerging discipline of neuroscience, and the work of the Allen Institute suggests transcriptomics of neuropeptides and corresponding receptors may characterize cell types. We find that DMV neurons are not solely cholinergic, but simultaneously catecholaminergic and GABAergic, including those that project to the heart. These findings challenge the conventional neuronal nomenclature. We propose that inputs determine phenotype, and work done on the Mouse Brain Atlas supports this premise, suggesting a transcriptomic diversification, less region‐specific, results from interaction with the environment. We present here a characterization of DMV cell types by input‐output transcriptomics. We combine high throughput transcriptomics, 3D anatomical mapping, and neural tracing in 12 week old Sprague Dawley male and female rats to interrogate the phenotypes of DMV neurons. To distinguish potential mechanisms of cardioprotection, we highlight the functional significance of several transcripts for secreted proteins in cardiac‐projecting DMV neurons. We have spatially mapped the location and molecular phenotypes of these single cells in the DMV and Nucleus Ambiguus (NA). The cardiac‐projecting neurons of the DMV extend approximately 2.5 mm rostrocaudally. The cardiac‐projecting neurons of the left DMV are particularly prominent caudally, while the cardiac‐projecting neurons of the right DMV are particularly prominent rostrally. A unique phenotype, with transcriptomic markers of strong cardioprotective potential, is present at the rostrocaudal position of left DMV previously shown to affect ventricular contractility. There is consistency between our findings from three spatial transcriptomics techniques. Based on these results, we propose a 3D anatomical and molecular map of cardiac vagal motoneurons.