bluuebunny/crossref_metadata_2025 huggingface dataset:
Aromatic C‐F Interactions Influence Binding Mode of Inhibitors in HDAC6
Authors: Watson Paris R. , Hooker Jacob , Christianson David W.
Published: May 2022

Fluorine is increasingly being incorporated into pharmaceuticals to modulate their physical properties. Furthermore, 18F can be utilized in positron emission tomography (PET) probes for in vivo imaging. Bavarostat is a selective inhibitor of histone deacetylase 6 (HDAC6), and when derivatized with 18F is used to map out HDAC6 localisation within the brain. As HDAC6 is implicated in Alzheimer’s disease due to its function as a Tau deacetylase, it may serve as an effective biomarker for this disease. The active site cavity of HDAC6 contains two phenylalanine residues that form an aromatic crevice, F583 and F643, that engage in offset π‐π interactions with phenylhydroxymate binding groups. Moreover, the fluoroaromatic ring of Bavarostat orientates such that the C–F group binds in the aromatic crevice instead of being exposed to solvent. Structure activity relationships were determined for a series of fluorophenylhydroxymates to inform the design of fluorinated inhibitors of HDAC6 as well PET probes. Our studies show the aromatic crevice of HDAC6 preferentially binds C–F groups in this series of inhibitors. The inhibitors show differences in zinc binding orientations, likely due to the electronic influence of fluorine on the hydroxymate zinc‐binding group.
Small extracellular vesicles induced gene expression changes in microglia
Authors: Luo Xuan , Jean‐Toussaint Renée , Sacan Ahmet , Ajit Seena
Published: May 2022

Background

Small extracellular vesicles (sEVs) are 30‐150 nm membranous particles released by a variety of cells and serve as a mediator in intercellular communication between adjacent and distal cells. sEVs carry biomolecular cargo including miRNAs, mRNAs, proteins, and lipids, which are selectively sorted and packaged and mirror the physiological state of the donor cells. Disease states can alter sEV composition affecting the message carried and thereby, its functional impact. Microglia, as the tissue‐resident macrophages and primary innate immune cells of the central nervous system, play an important role in neuropathic pain. Here, we investigated alterations in the composition of serum sEVs from a mouse model of neuropathic pain and assessed the functional consequences of sEV uptake by primary cortical microglia.

Methods

sEVs were purified from the serum of a spared nerve injury (SNI) mouse model of neuropathic pain and sham‐operated mice four weeks after surgery. sEV uptake by primary cortical microglia was confirmed by incubating microglia with 1 µg of PKH26‐labeled sEVs for 24 h and visualized using confocal microscopy. Transcriptome and cytokine alterations induced by sEV uptake in microglia (1 µg for 24 h) were determined using RNA‐seq and cytokine array.

Results

We identified a differential expression of 22 miRNAs in serum sEVs derived from SNI and sham control mice. Confocal microscopy confirmed the presence of sEVs in microglia following 24 h sEV incubation. We observed a significant difference in gene expression resulting from the uptake of distinct serum sEVs – alterations were more prominent in microglia treated with sham sEVs. Gene Ontology analysis indicated gene expression changes induced by the uptake of sEVs from both SNI and sham surgery were both implicated in immune responses, but genes upregulated by sham sEVs were also enriched in inflammatory responses. Secretions of pro‐inflammatory cytokines and chemokines including CXCL10, CXCL2, and TNF‐α were higher in microglia incubated with sEVs from mice that had sham surgery compared to PBS control or SNI sEVs.

Conclusions

Our data demonstrate alterations in sEV composition from both SNI and sham surgeries, and the molecular changes captured in donor sEVs could impact gene expression in the recipient cells. The differences observed in microglial phenotypes and gene expression suggest sEVs from sham surgery are directed at wound healing and thus differ from nerve injury‐induced sEVs.
Endocannabinoid regulation of behavior in response to negative affective states associated with alcohol abstinence
Authors: Tat Jennifer , Thibeault Kimberly C. , Adank Danielle N. , Centanni Samuel W. , Winder Danny G. , Calipari Erin
Published: May 2022

Alcohol use disorder (AUD) is characterized by elevated levels of alcohol intake followed by negative affective states when individuals halt alcohol consumption. In individuals suffering from AUD, alcohol taking and seeking is often hypothesized to be motivated by negative reinforcement, where individuals continue consuming alcohol in order to avoid the negative affective states that are triggered by abstinence. Females are particularly vulnerable to the effects of AUD and are more likely to drink to regulate this negative affect produced by abstinence. The endogenous cannabinoid (eCB) signaling system is heavily implicated in the mediation of both reward and reinforcement, and alcohol consumption has been shown to be positively correlated with release of the eCB ligand 2‐arachidonoylglycerol (2‐AG) within the reward neurocircuitry. Taking this into consideration, we took a multifaceted approach to investigate the mechanisms by which individuals avoid negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by prolonged forced abstinence (CDFA) and pharmacological enhancement of eCB with JZL184, we were able to examine how altered eCB signaling can affect how females respond to avoid negative outcomes. First, by using negative reinforcement operant conditioning, we show that increased eCB signaling via a single injection of JZL184 causes an increase in acquisition rate as compared to ethanol‐treated and control animals. After reaching acquisition, a difference in behavioral pattern is noted; when given the chance to either escape or avoid the foot shock, JZL‐treated animals consistently chose to escape the shock rather than fully avoid it, possibly indicating a reduced sensitivity to the aversive stimulus. Next, when comparing latency to first bite in the novelty‐suppressed feeding test (NSFT), a common protocol used to measure depression and anxiety, with acquisition rate, we found that, in ethanol‐treated animals, decreased latency to eat is negatively correlated with days to acquisition. Moreover, we are able to reverse this effect with a single dose of JZL184. Together, these data indicate that a single dose of JZL184 has the ability to cause long‐lasting effects on the eCB signaling system and, consequently, the reward circuitry. Moving forward it will be critical to directly link these effects between reinforcement learning and reward processing in order to develop a pharmacological intervention to reduce the negative affective states following alcohol abstinence.
Hesperetin modulation of oxidative stress and inflammatory mediators in LPS‐Activated BV‐2 Microglial Cells
Authors: Evans Jasmine , Mendonca Patricia , Soliman Karam F. A.
Published: May 2022

Neurodegeneration affects millions of Americans each year, occurring in later periods of life, where its onset is induced by neuroinflammation and oxidative stress. Flavonoids are known to protect against damages caused by reactive oxygen species (ROS) and reactive nitrogen species (RNS). This study evaluated the effect of hesperetin (HST), a major flavonoid found in oranges and mandarins, on LPS‐activated BV‐2 microglial cells. The obtained results of RT‐PCR showed that HST down‐regulated the mRNA expression of PD‐L1 in more than 50% of the LPS‐activated cells. In addition, HST induced the mRNA expression of Nrf2, which is involved in the transcription of several antioxidant genes. Moreover, the data from the oxidative stress PCR arrays showed that after 24 h, the concentration of 100 µM of HPT modulated numerous genes that regulate oxidative stress and inflammatory processes. HST down‐regulated the mRNA expression of ERCC6, ACTB, SQSTM11, NOS2, and NCF1 and up‐regulated the expression of HMOX1, which participate not only in excessive oxidative stress processes, by also in exacerbated inflammatory states. Both CAT and SOD enzymatic assays showed that treatment with HSP and LPS alone had no effect on CAT expression, but when cells were treated with the combination of HST and LPS, there was a reduction in these expressions enzymes. The quantitative glutathione assays demonstrated that only the combination treatment of HST and LPS reduced the expression of glutathione after a 24‐h treatment period. In conclusion, these data show that hesperetin modulates several genes associated with oxidative stress and neuroinflammation and may potentially slow the progression of neurodegeneration.
Effects of Cardamonin on PD‐1/PD‐L1 Checkpoint in Triple‐Negative Breast Cancer
Authors: Mendonca Patricia , Hill Lonnie , Soliman Karam F. A.
Published: May 2022

Breast cancer is the most common type of cancer in women and the second leading cause of death. Trible‐negative breast cancer (TNBC) accounts for about 10% of all breast cancer. Unlike other forms of breast cancer, TNBC does not express estrogen, progesterone, or HER2 receptors. These cancer cells instead employ mechanisms to escape the immune system. One such mechanism involves the upregulation of the expression of PD‐L1, a ligand encoded by the CD274 gene. Because cancer cells tend to produce higher levels of PD‐L1 compared to normal cells, therapies that inhibit PD‐L1 may be helpful. Flavonoids, polyphenols found abundantly in fruits and vegetables, have already been shown to be effective antioxidants, anti‐inflammatories, and anticancer. Cardamonin, an aromatic enone within the flavonoid family, has been shown to display an array of pharmacological activities. The objective of this work was to investigate the ability of Cardamonin in decreasing the expression of PD‐L1 in MDA‐MB‐231 (Caucasian) and MDA‐MB‐468 (African American) TNBC cell lines which are genetically distinct. The methodology included cytotoxicity assays, human B7H1/PD‐L1 ELISA, and Real Time‐Polymerase Chain Reaction (RT‐PCR) assays. The results obtained showed that cardamonin treatment caused a dose‐dependent decrease in cell viability in both cell lines in concentrations ranging from 3.12 µM to 200µM. ELISA data showed low levels of PD‐L1 expression in both cell lines. Furthermore, RT‐PCR assays demonstrated that cardamonin downregulated PD‐L1 expression in MDA‐MB‐231cells in the presence or absence of IFN‐γ, showing a more effective effect after the cells were stimulated. In MDA‐MB‐468 cells, cardamonin had an opposite effect, upregulating the expression of PD‐L1. The data show that cardamonin may alter the production of PD‐L1 at the transcription level in MDA‐MB‐231 TNBC cells, but not in MDA‐MB‐468 cells, corroborating with literature showing differences in the tumor microenvironments between African Americans and non‐African Americans. In conclusion, cardamonin may have the therapeutic potential to decrease the levels of PD‐L1 in the tumor microenvironment combating the cancer cells’ effectiveness of evading the immune system.
Effects of Hyperoxia on Coronary Circulation in Patients with Peripheral Artery Diseases (PAD)
Authors: Gao Zhaohui , Stavres Jonathon , Brandt Kristen , Blaha Cheryl , Cauffman Aimee , Luck J. C. , Sinoway Lawrence , Cui Jian
Published: May 2022

It is well‐known that hyperoxia evokes different effects in patients with different dieaseas conditions. Studies from this lab and others have shown that hyperoxia is a potent coronary vasoconstrictor. Recently, we found that hyperoxia exaggerates systolic blood pressure (BP) response in patients with PAD. The aim of this study is to characterize the coronary circulatory effects of hyperoxia in PAD patients and healthy controls. We recruited 20 PAD patients (14M, 6F, 66 ± 6yrs), and 16 age‐matched control subjects (10M, 6F, 65 ± 8yrs). The ankle‐brachial pressure index (ABI) in the most affected limbs of the patients was 0.58 ± 0.15, compared to the controls ABI of 1.04 ± 0.09. For the hyperoxia procedure the subjects breathed room air for three minutes and 100% oxygen for five minutes. BP and heart rate (HR) were monitored during baseline and hyperoxia. The rate pressure product (RPP), an index of myocardial oxygen consumption, was calculated as the product of systolic BP and HR. Transthoracic Coronary Echocardiography was used to record early and late peak coronary blood flow velocities (CBV1 and CBV2) in the left anterior descending artery. Early and late coronary vascular conductances (peak coronary blood velocity/diastolic blood pressure, CVC1 and CVC2) and coronary perfusion time fraction (CPTF, coronary perfusion time/R‐R interval) were calculated. All data was compared across time and between groups using Repeated Measures Analyses of Variance (RMANOVA).

Results

At the baseline, RPP and coronary parameters (CBV2, CVC1, and CVC2) were higher in the PAD group compared to controls, also the PAD group showed significantly shorter CPTF. During hyperoxia, CBV1, CBV2, CVC1, and CVC2 decreased to varying degrees in both groups. However, no significant interactions were observed for RPP and all coronary parameters during hyperoxia between the groups (Table). Our findings suggest that the effect of hyperoxia on coronary circulation was not significantly different between PAD and healthy controls.
Engineered Nuclear Import Receptor Karyopherin‐β2 Chaperones Aberrant Phase Transitions of Disease‐Associated Cargo
Authors: Fare Charlotte M. , Korobeynikov Vladislav A. , Rhine Kevin , Yoniles Joey , Myong Sua , Shneider Neil A. , Shorter James
Published: May 2022

Karyopherin‐β2 (Kapβ2) is a nuclear import receptor (NIR) that localizes proteins bearing a proline‐tyrosine nuclear localization signal (PY‐NLS) to the nucleus, including the RNA‐binding protein (RBP) FUS. Recent work has demonstrated that NIRs can chaperone RBPs in vitro and in cells, preventing and reversing their self‐assembly and aggregation. However, when the PY‐NLS is mutated, Kapβ2 no longer efficiently chaperones its cargo. Here, we focus on the case of FUSP525L, an RBP NLS mutant associated with a highly aggressive form of juvenile ALS. Using a structural approach, we have engineered Kapβ2 to recognize mutant FUS and demonstrate improved chaperone activity in vitro, in human cells, and in mice.
Understanding the Development of Atherosclerosis: Computational Model Depicting the Interaction Between Low‐Density Lipoproteins and Vascular Calcification
Authors: Kosciuszek Nina D. , Megalaa Gorg , Savinova Olga V.
Published: May 2022

Objective

Cardiovascular disease (CVD) affects many individuals in the United States each year. It has many modifiable risk factors, such as hyperlipidemia and hypertension. Another risk factor is vascular calcification, which has been shown to be an independent risk factor in mortality in patients with CVD. Vascular calcification and the number of circulating low‐density lipoprotein (LDL) particles influence atherosclerotic plaque formation and progression. However, the interaction between calcification and atherosclerosis is not completely understood.

Hypothesis

We have previously shown that vascular calcification modifies the endothelial surface topology, thus altering the amount of wall shear stress (WSS) present against the arterial wall. Therefore, we hypothesize that more LDL particles will approximate near the arterial wall, specifically in areas exhibiting low WSS. We postulate that vascular calcification influences LDL particle accumulation near the arterial wall, hence contributing to the pathogenesis of atherosclerosis.

Methods

As previously reported, we created a Fluid Structure Interaction (FSI) model from abdominal aorta geometries that were reconstructed from micro‐CT images at 5.7 µm resolution. One geometry was from mico‐CT images of a mouse that were genetically modified to have microcalcifications within the subendothelial layer (affected). In contrast, a mouse that was not genetically modified served as the control aorta geometry (control). This FSI model considered a nonlinear structure and hyperplastic materials in a steady‐state phase. A Particle Tracing module was added to this model in COMSOL Multiphysics 5.6 (COMSOL Inc.). This allowed for the examination of the behavior of 2,100 LDL particles in relation to the rough and smooth surfaces of the affected and control aortas.

Results

The larger curvature of the affected aorta contained the greatest quantity of calcified lesions. This curvature demonstrated more LDL particles accumulating near the interior wall compared to the larger curvature of a healthy aorta. It was observed that the aorta with the calcified nodules experience a lower magnitude of WSS compared to the control aorta. It was observed that the particles clustered in areas that exhibited low WSS, ranging from less than 10 N/m2in the affected aorta.

Conclusions

Rougher surface topology of a calcified aorta illustrates a positive correlation with LDL particle accumulation in the areas of low WSS. This provides insight to how WSS and vascular calcification influence atherosclerotic plaque formation and progression based on the behavior of LDL particles in this model. Further work needs to be done to explore other material properties to better simulate and understand this interaction.
Synthesis of Novel Tubulin Inhibitor Candidates as Potential Anti‐Cancer Agents
Authors: Faulkner Grace , Pelkey Erin
Published: May 2022

Tubulin polymerization and depolymerization are vital for cellular processes, such as cell division, making tubulin inhibition a viable target for the development of anti‐cancer agents. Previous studies have confirmed that an indole‐containing heterocycle we synthesized exhibits anti‐cancer activity through a tubulin polymerization inhibitory mechanism. The heterocycle is structurally related to combretastatin A4, and is thought to interact with tubulin at the colchicine binding site. Our goal is to build upon the existing library of indole‐containing heterocyclic analogs and synthesize novel tubulin inhibitors with increased potency. Molecular docking studies with the colchicine binding site of tubulin informed the design of PY‐391‐Das a tubulin inhibitor candidate. Thus, we are working toward synthesizing this novel compound using modular synthetic strategies. Our synthetic strategy encompasses five reactions. Three out of the five synthetic steps toward PY‐391‐Dhave been completed in high yield. Two reactions that have been previously optimized by our research group have been applied to novel compounds en route to PY‐391‐D, demonstrating the wide applicability of our synthetic strategies. The optimization of the butyl‐group addition to the indole‐nitrogen through an SN2 mechanism was investigated in this study, and has been applied to other electrophiles. Reaction success has been monitored through compound characterization facilitated by 1H NMR spectroscopy analysis. The progress towards completing the streamlined synthesis to produce tubulin inhibition candidates, including PY‐391‐Dand structurally related analogs, are detailed in this study.
The Identification of NIFK’s RNA Binding Targets and Its Prognostic Values in Lung Cancer
Authors: Lin Tsung‐Chieh
Published: May 2022

Background

We previously identified nucleolar protein interacting with the FHA domain of pKi‐67 (NIFK) as a poor prognostic marker in lung cancer. Furthermore, NIFK overexpression promotes cancer metastasis via decreasing casein kinase 1α (CK1α) expression, a suppressor of pro‐metastatic TCF4/β‐catenin signaling. We further observe the axis is induced by the downregulation of a novel transcription factor RUNX1. It is also known for NIFK’s biological function in regulating RNA metabolism via RNA recognition motif (RRM). However, a comprehensive analysis for identifying the RNA binding targets has not yet been reported. In this study, we aim to unravel the interactive mechanism and to characterize its potential clinical value in cancer patient cohort.

Method

NIFK is pointed out to possess RNA binding function via RRM. RNA immunoprecipitation assay was performed using specific NIFK antibody in lung cancer PC13 and A549 cells after NIFK overexpression. RNA targets were analyzed by RT‐PCR and Next Generation Sequencing (NGS). In addition, expression levels were investigated by IHC staining and omics data mining. The association of the respective expression with patient survival outcome were explored in lung cancer cohorts.

Results

We observe the increase in amount of RNA binding targets after NIFK overexpression respectively in PC13 and A549 cells as comparing with the control. Moreover, NIFK appears to bind with RUNX1 RNA but not CK1α suggesting it is the initial step of NIFK‐mediated TCF4/β‐catenin signaling regulation. NIFK also leads to RUNX1 RNA instability of which mechanism remains to be explored. RUNX1 level is observed to associate with good prognosis in lung cancer datasets (jacob‐00182‐CANDF and GSE13213). Furthermore, direct interactive RNA targets of NIFK are identified by RNA immunoprecipitation and RNA‐Seq, which indicate SYNE2 as one of targets with prognostic significance in lung cancer. Increased SYNE2 is detected upon NIFK overexpression, and the lung cancer patients stratified in high SYNE2 group appear to associate with inferior clinical outcomes.

Conclusion

We first show the interactive mechanism of pro‐metastatic molecule NIFK in regulating TCF4/β‐catenin signaling and SYNE2, which might provide insights into therapeutic development for lung cancer patients.
Scleraxis is Required for Induction of GLS1 Expression in Cardiac Myofibroblasts
Authors: Chattopadhyaya Sikta , Nagalingam Raghu S. , Narhan Pavit , Ledingham Dayna A. , Moffatt Teri L. , Czubryt Michael P.
Published: May 2022

Cardiac fibrosis is an aberrant wound healing process involving excessive production of extracellular matrix proteins resulting in impaired systolic and diastolic function of the heart, contributing to heart failure and death. Glutaminolysis, which involves conversion of glutamine to glutamate by glutaminase (GLS), and conversion of glutamate to α‐ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, has been reported as a mechanism for meeting the energy demand required for pulmonary and liver fibrosis however its contribution in cardiac fibrosis is unclear. Although the role of the transcription factor Scleraxis has been established in the phenotypic conversion of cardiac fibroblasts to fibrotic myofibroblasts by our lab, Scleraxis’ role in glutaminolysis remains unelucidated. Our results here show Scleraxis expression was increased by 9 fold and GLS1 by 4 fold when comparing freshly isolated rat cardiac fibroblasts to fibrogenic myofibroblasts. While Scleraxis overexpression increased GLS1 expression 20‐fold, Scleraxis knockdown attenuated GLS1 expression by 76% in cardiac myofibroblasts while cardiac fibroblasts from Scleraxis knockout mice exhibited a 90% loss of GLS1 expression. TGFβ, one of the primary inducers of cardiac fibrosis induces enhanced expression of Scleraxis and has been found to be highly dependent on Scleraxis for many of its downstream effects. TGFβ treatment of activated cardiac fibroblasts from wild type mice doubled the expression of GLS1, but failed to induce GLS1 expression in Scleraxis knockout cells. Via luciferase assay we found a dose‐dependent transactivation of the human GLS1 promoter by Scleraxis in NIH3T3 fibroblasts. While Scleraxis significantly transactivated the hGLS1 promoter, mutation of an E‐Box Scleraxis putative binding site caused 70% attenuation of promoter transactivation. These findings suggest that Scleraxis is sufficient and required to regulate GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.
Lamin A/C Downregulation Alters Prostate Cancer Cellular Phenotypes
Authors: salus Griffin J. , Strojanova Marija , Heaphy Christopher M.
Published: May 2022

Nuclear lamina proteins are associated with the structural stability of cells and the anchoring and localization of heterochromatin. The downregulation of lamin A/C has been shown to contribute to cellular deformity, genome instability, and affect the ability of cells to differentiate. Therefore, evaluating lamins in prostate cancer progression may uncover important mechanisms for coordinated regulation of cellular processes, thereby highlighting potential vulnerabilities that may be targeted therapeutically to alter the clinical course of the disease. Previous multiplex fluorescent immunolabeling experiments showed a substantial fraction of prostate cancers displaying dramatically diminished lamin A/C expression in the cancer cells compared to the benign prostate epithelial (luminal and basal) cell populations. In this study, we investigate how downregulation of lamin A/C directly affects cellular characteristics associated with metastasis. By knocking down lamin A/C expression (~50%) with siRNAs targeting lamin A/C, we tested how the modulation of lamin A/C directly affected cell proliferation, clonogenic survival, and cell invasion in three prostate cancer cell lines (PC3, DU145, and CWR22Rv1). Compared to a scrambled siRNA control, all three cell lines showed no significant change in proliferation and clonogenic survival when lamin A/C was downregulated. While PC3 and DU145 showed no significant change in invasion potential, lamin A/C knockdown in CWR22Rv1 resulted in a decreased invasion potential of 22.8% compared to controls. We are conducting further experiments to fully elucidate the effects of lamin A/C downregulation on these metastasis‐relevant cellular properties. Overall, these findings will help to further understand the significance of alterations in the nuclear lamina protein's involvement in metastatic progression, and to further validate lamin dysregulation measurements as a potential biomarker to predict clinical outcomes in prostate cancer.
Selection of culture media for chicken PGC long‐term cultivation
Authors: Barkova Olga , Larkina Tatyana , Krutikova Anna , Peglivanyan Grigoriy , Polteva Ekaterina
Published: May 2022

Primordial germ cells (PGCs) are precursors of eggs and spermatozoa and serve as an effective means for the transgenic chickens production, including using CRISPR / Cas9 technology. The aim study is selection of PGCs culture media suitable for long‐term cultivation. All experiments on chickens were conducted with ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding ‐ Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020‐3). For the work, eggs of the Pushkin breed chickens (n = 21) were selected from the Bioresource collection RRIFAGB (Pushkin, St. Petersburg). PGCs were selected from the embryos dorsal aorta for 3‐4 days of incubation (stage Hamburger‐Hamilton 14) using a microinjector (Narishige IM‐11‐2, Japan). Cells were cultured for 21 days at 37 ° C with 5% CO2. Three variants of the culture medium were used. The first basic medium was Opti‐MEM (Reduced Serum Medium, GlutaMAX Supplement) (Gibco, Thermo Fisher) with the addition of sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl. On day 21, the concentration of cells in the first culture reached 1,2х103, and some of the wells with cells were overgrown. The second base medium was similar to the first base medium, but with the addition of antimycotic antibiotic (Thermo Fisher) up to 1X. The medium with the use of the antibiotic‐antimycotic was less susceptible to overgrowth, on day 21, the concentration of cells in the culture reached 3.5x104. The third basic medium was KnockOut DMEM / F‐12, without L‐glutamine (Gibco, ThermoFisher) with the addition sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Human Activin A Recombinant Protein (Gibco, Thermo Fisher) ‐ 25 ng / μl, Human FGF‐basic (FGF‐2 / bFGF) Recombinant Protein (Gibco, Thermo Fisher) ‐ 10 ng / μl, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl), antimycotic antibiotic (Thermo Fisher) up to 1X. The third medium with the addition of Activin A and FGF‐2 / bFGF supported the cell culture better, overgrowth was practically not observed, cells were actively proliferating, оn day 21, the concentration of cells in the culture reached 2.7x105. The PGCs presence was confirmed by expression high level (p≤0.01) specific marker genes PGCs such as CXCR4 (chemokine receptor CXC type 4) and PIWIL1 (Piwi‐like protein 1) in cultured cells. In a control tissue small intestine sample of an adult chicken (330 days old), the above genes expression was completely absent.
A multiplex model in a Drosophilaidentifies novel gene‐environment interactions: A step towards personalized medicine
Authors: Sarkar Souvarish
Published: May 2022

Epidemiological studies strongly support a role for environmental factors, particularly pesticide exposure, in the pathogenesis of Parkinson's disease (PD). Genetic risk factors, including penetrant single‐gene mutations and risk factors identified from genome‐wide associated studies (GWAS), also contribute to PD risk and progression, but available model systems are limited in their ability to interrogate gene‐environment crosstalk in vivo and at scale. Here we use Drosophila as a model system to understand complex gene‐environment interactions in PD. We developed a multiplex model in Drosophilain which we knocked down GWAS candidate genes in neurons in a robust new α‐synucleinopathy model and exposed the flies to environmental neurotoxicants. We identified an interaction between LRRK2, rotenone, and α‐synuclein. Expression of the disease‐causing Lrrk‐G2019S mutant in the presence of rotenone and alpha‐synuclein induced behavioral deficits and mitochondrial dysfunction. Further, super‐resolution microscopy analysis revealed that the interaction of LRRK2, α‐synuclein, and rotenone leads to hyperstabilization of the actin cytoskeleton. We have previously shown that LRRK2 has actin severing activity, and previous studies have implicated the GTPase domain in regulating actin severing. We next expressed a GTPase domain mutant, Lrrk‐Q1003H, designed to mimic the human protective mutant LRRK2‐R1398H. Interestingly, expression of the LRRK2 protective mutant attenuated behavioral deficits mediated by LRRK2‐rotenone‐synuclein interactions. Further, the expression of the protective mutant also attenuated the actin stabilization and mitochondrial deficits. Moreover, genetic analysis from a patient cohort who had previous pesticide exposure revealed that the patient's LRRK2‐R1398H mutation reduces the chance of developing PD. Since global actin severing may have unwanted side effects, we used a combination of forward genetic screen and proteomics to identify potential kinases that can be druggable targets. We identified Cdc42 binding protein kinase MRCKα, an actin‐binding protein, as a potential target. Genetic and pharmacological inhibition of MRCKα in Drosophila can modify the toxicity induced by the interaction among LRRK2, α‐synuclein, and rotenone by inhibiting actin hyperstabilization. Using our novel multiplex model in Drosophila, we have identified an interaction between LRRK2, α‐synuclein, and rotenone which is modulated by actin stabilization and mitochondrial dysfunction. We have further demonstrated that using this multiplex approach, we can study the mechanism of these interactions as well as identify novel drug targets for the interactions. Our findings have implications towards the development of a personalized approach for drug discovery and lead identification.
PINK1‐Mediated Neuroprotection Against Mutant Tau Pathology
Authors: Lizama Britney N. , Wang Kent Z. , Verma Manish , Zhu Jianhui , Skeel Abigail , Chu Charleen T.
Published: May 2022

Protein accumulation, mislocalization and dendritic atrophy are pathological features of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Frontotemporal lobar dementia (FTLD). Postmortem samples from patients with AD or FTLD reveal Tau pathology, wherein aberrant Tau accumulates as oligomers and hyperphosphorylated species. Our preliminary data show that mutant Tau (Tau‐A152T), which is linked to increased risk of AD and FTLD, causes dendritic simplification in cultured neurons. While aberrant Tau accumulation is linked to disease severity in AD and FTLD, endogenous factors that can prevent Tau toxicity remain unknown. We and others have shown that PTEN‐induced Kinase 1 (PINK1) is protective in a wide range of genetic and toxin‐based models of neurodegeneration. Elevating PINK1 levels in vitro enhances dendrite length, while loss of PINK1 causes dendrite simplification. Moreover, using our own cohort of AD, FTD and 4R‐tauopathies, we confirmed published observations of decreased PINK1 mRNA and protein expression in the brains of patients with AD. In this work, we test the potential of elevating PINK1 to protect against mutant Tau pathology. We hypothesize that elevated PINK1 expression can protect against dendrite simplification caused by mutant Tau. To test this hypothesis, mouse primary cortical neurons and cortical neurons differentiated from control human iPSC lines (ic‐neurons) were transfected with GFP only or co‐transfected with Tau‐A152T. To elevate PINK1 levels, cultures were co‐transfected with PINK1‐GFP or treated with a pharmacological inhibitor of PINK1 degradation (BC1464). Through neurite length and Sholl analysis, we found that increased PINK1 levels protects neuronal cultures against mutant Tau‐mediated dendritic simplification. To determine the mechanism of PINK1‐mediated protection, we sought to test whether PINK1 elevation promotes Tau clearance. SH‐SY5Y cells were transfected with Tau‐A152T and treated with either DMSO (vehicle) or BC1464. A six‐hour cycloheximide chase assay was performed, and we found that Tau‐A152T levels decreased with BC1464 treatment, but not in vehicle‐treated cultures. Endogenous, wild type Tau levels were not significantly affected. We recently discovered that PINK1 interacts with Valosin‐Containing Protein (VCP, p97), a critical regulator of proteostasis pathways, to modulate dendritic morphology. Ongoing work is aimed at studying the underlying mechanism(s) of PINK1‐mediated Tau‐A152T clearance and identify the signaling partners with which PINK1 interacts to promote neuroprotection.
Dissociation of hypertension and renal damage after cessation of high salt diet in Dahl rats
Authors: Arkhipov Sergey N. , Liao Tang‐Dong S. , Potter D'Anna L. , Bobbitt Kevin R. , Ortiz Pablo A. , Pavlov Tengis S.
Published: May 2022

Background

Every year, thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. Most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying the self‐sustaining character of hypertension. We hypothesize that chronic hypertension leads to sustained renal damage and excessive sodium reabsorption, staying even after removing the trigger, caused the initial blood pressure raise.

Methods

We used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4%NaCl diet to induce hypertension, and then the diet was switched back to normal (0.4%NaCl).

Patch‐clamp analysis was performed in freshly isolated, split‐open cortical collecting ducts to characterize activity of epithelial sodium channel (ENaC), responsible for sodium reabsorption in aldosterone‐sensitive distal nephron. Flow cytometry, trichrome staining and FITC‐inulin clearance estimated immune cell infiltration, renal damage and renal function.

Results

We found that blood pressure increase is reversible if induced by a short 3‐day long feeding with 4%NaCl diet. A 3‐week long high salt diet develops a sustaining hypertension (MAP ~150 mmHg): switching back to 0.4%NaCl diet slightly reduces blood pressure (to 145 mmHg) next day, but later hypertension progresses, reaching ~158 mmHg. Notably, the self‐sustaining phase of hypertension was sensitive to benzamil treatment, which lowered BP to ~136 mmHg.

Patch clamp analysis revealed that development of hypertension was accompanied with elevated ENaC activity which also stayed high, despite withdrawal of 4%NaCl diet.

A separate subset of experiments demonstrated that dietary salt reduction in hypertensive animals decreases the number of total CD45+CD3+CD4+ and CD45+CD3+CD8+ cells in renal tissues.

Also, we found a reduced area of protein casts abundance in the post‐salt hypertensive group in comparison with age‐matched rats kept on a high salt diet, and improving a glomerular filtration rate in the post‐salt period.

Conclusion

Based on our earlier publications and current data, we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge. However, dietary salt restriction reduces renal inflammation and damage in hypertensive animals.
Impact of social environment on alcohol‐induced behavior
Authors: Murillo Gonzalez Dilean J. , Alvarado Jose , Hernandez Bryan , Sabandal Paul , Han Kyung‐An
Published: May 2022

Alcohol consumption occurring in social or solitary settings often yield different behavioral responses. For example, drinking of alcoholic beverages in a social setting is associated with enhanced positive effects including euphoria and increased sociality while drinking in a solitary setting is linked to augmented negative effects such as depressive symptoms and anxiety. The neurobiological mechanism by which the social environment impacts on behavioral responses to alcohol, however, remains understudied. Using the Drosophilamodel, we investigated whether distinct social environments affect behavioral responses to ethanol. In this study, we focused on the hyper‐locomotor activity that is typically used as a proxy for euphoria induced by ethanol. Wild‐type Canton‐S (CS) flies were exposed to ethanol in either socially‐isolated (1 fly per chamber) or socially‐enriched (13 flies per chamber) settings and their locomotor activities were analyzed. We found that the socially‐enriched flies showed significantly more hyperactivity under the influence of ethanol compared to the singly exposed flies. This suggests that the presence of other flies amplifies ethanol‐induced euphoria. Dopamine is known to be important for social interactions thus we explored the role of dopamine in the social effect on the ethanol response by examining the dopamine transporter mutant fumin with hyperdopaminergic tone. When exposed to ethanol in a social setting, fuminmutants showed the augmented hyperactivity compared to the singly exposed fumin as well as single‐ and group‐ exposed CSflies. Together, these findings suggest that hyperdopaminergic signaling augments the effect of social enrichment on the ethanol response. The findings of this study provide important information regarding the mechanism by which genetic (dopamine transporter) and non‐genetic (social context) factors interact for the locomotor response to ethanol.
Altered Adipose Tissue Inflammatory Markers in Mothers With Gestational Diabetes
Authors: Maloney Alan , Barnas Jillian , Vieira‐Potter Victoria , Kanaley Jill
Published: May 2022

INTRODUCTION

Adipose tissue (AT) releases adipokines and inflammatory cytokines which may have an impact on the fetus during pregnancy. Mothers with gestational diabetes (GDM) have children who have an increased risk of developing obesity and diabetes compared to children born to normal glucose tolerant (NGT) mothers, but the mechanisms are not known. AT dysfunction in mothers with GDM may play a mechanistic role in the relationship.

HYPOTHESIS

Mothers with GDM will have impaired AT mitochondrial function and increased markers of inflammation.

METHODS

AT tissue samples were collected from 22 NGT and 6 GDM pregnant women undergoing a C‐section. Subcutaneous (SQ) and visceral AT (VAT) samples, mother's blood, and fetal cord blood were collected. AT samples were assessed for protein expression (via Western blot) of EGF‐like module‐containing mucin‐like hormone receptor‐like 1; estrogen receptors (ERα and ERβ); adiponectin; mitochondrial oxidative phosphorylation (Ox‐phos) C1‐5; nuclear factor kappa‐light‐chain‐enhancer of activated B cells; adipose triglyceride lipase (ATGL) and a panel of inflammatory, mitochondrial, and metabolic genes via quantitative real time polymerase chain reaction. Mother’s blood and cord blood were assessed for presence of adiponectin, brain derived neurotrophic factor, C‐reactive protein. and non‐esterified fatty acids via enzyme‐linked immunoassay.

RESULTS

In GDM compared to NGT mothers, VAT had significantly lower protein expression levels of Ox‐phos C2 and adiponectin (p=0.009 and 0.008 respectively) and trended towards significance for ATGL and Ox‐phos C3 (p=0.05 and 0.06 respectively). Gene expression of adiponectin and ERβ also tended to be lower in GDM mothers (p=0.05 and 0.06 respectively). In SQAT, higher gene expression of mannose receptor C‐type 1 (anti‐inflammatory macrophage marker) in the GDM mothers was found (p=0.037). No differences in the measured blood markers were found.

CONCLUSION

Data support that mothers with GDM differentially express AT adipokines and genes associated with inflammation, insulin resistance and altered lipid metabolism than mothers with NGT.
TLK1‐MK5 Axis Modulates Actin Filaments and Focal Adhesion Components to Promote PCa Cell Migration and Invasion
Authors: Khalil Md Imtiaz , Singh Vibha , King Judy A. , De Benedetti Arrigo
Published: May 2022

Background

Metastatic dissemination of prostate cancer (PCa) accounts for majority of PCa related deaths. However, the exact mechanism of PCa cell spread is still unknown. We uncovered a novel interaction between two unrelated promotility factors, tousled‐like kinase 1 (TLK1) and MAPK‐activated protein kinase 5 (MK5), which initiates a signaling cascade promoting metastasis. In PCa, TLK1‐MK5 signaling might be crucial as androgen deprivation therapy (ADT) leads to increased expression of both TLK1 and MK5 in metastatic patients. Moreover, TCGA analysis also revealed higher expression of both TLK1 and MK5 in metastatic tumors. Therefore, we hypothesize that TLK1‐MK5 axis promotes PCa cell migration and invasion through actin reorganization as well as focal adhesion proteins modification and disruption of this interaction may help to keep the tumor localized.

Methods and Results

We conducted scratch wound repair and proliferation assays to determine if TLK1 and MK5 can regulate motility and proliferation. Both genetic depletion and pharmacologic inhibition of TLK1 and MK5 resulted in reduced migration of both non‐malignant MEF and PCa cells without affecting cellular proliferation rate. Trans‐well invasion assays also demonstrated reduced invasion of MK5−/− MEF cells in Matrigel and other ECMs. However, TLK1 overexpression alone in the MK5−/− MEF cells did not increase the wound healing and invasion which suggested that TLK1 cannot enhance cellular migration and invasion in absence of MK5. We confirmed TLK1 binding of MK5 by co‐IP, His‐ and GST‐pull down assays and established that TLK1 phosphorylates MK5 on three residues (S160, S354, S386) that results in the catalytic activation of MK5. While western blotting (WB) detected substantial amount of pMK5 S354 and TLK1 in all major PCa cell lines, anti‐androgen treatment increases pMK5 S354 level in a dose‐dependent manner and pharmacologic inhibition of TLK1 reduces pMK5 S354 level in LNCaP cells, which suggest TLK1 as an authentic pMK5 S354 kinase. IHC analysis of TRAMP mice and human PCa TMA also revealed higher pMK5 S354 level in metastatic lesions compared to the low‐risk tumors. In addition, pMK5 S354 was found to colocalize with neuroendocrine marker chromogranin A, together of which may suggest pMK5 as a potential biomarker of PCa aggressiveness. The motility reduction of PCa cells upon MK5 inhibition is a consequence of impaired actin filamentation as we observed in LNCaP and PC3 cells. Our WB analysis demonstrated that MK5 inhibition by a repurposed inhibitor results in decreased FAK Y861, Paxillin Y118, HSP27 S78/82 phosphorylation, and ERK3 stabilization, which could lead to altered actin filamentation, focal adhesions, morphology, as well as motility and invasion.

Conclusion

Our data support that TLK1‐MK5 axis is functionally involved in driving PCa cell metastasis and clinical aggressiveness, hence, disruption of this axis may inhibit the metastatic capacity of PCa.
Early Interventions for Toxicant Ocular Injury May Need to be Toxicant‐Specific
Authors: Ruff Albert , Lehman John , Causey Robert , LaGrasta Christina , Gomez Amber , Koenig Jeffrey
Published: May 2022

Corneal injuries induced by various toxicants share similar pathologies such as corneal opacity and neovascularization. Several studies suggest that the molecular mechanisms that drive these delayed and progressive pathologies may converge at later time points. However, chemicals with different toxicological properties are likely to elicit different molecular responses early after exposure. These early responses may present opportunities for therapeutic intervention. Our objective was to study the molecular mechanisms of toxicant‐induced corneal injury, utilizing siRNA high throughput screening (HTS). Our hypothesis was that early molecular responses to each toxicant would be dissimilar and targets validated in vitro may be effective for treating toxicant injury. We selected ~3000 targets from preconfigured Dharmacon Human Druggable libraries for three toxicants: chloropicrin (CP), hydrogen fluoride (HF), and sulfur mustard (HD). Microarray data from injured mouse corneas were used to focus library selection. Screening was conducted in the human immortalized corneal epithelial cell line SV40‐HCEC. Endpoints included IL‐8 levels in cell culture supernatants and cell viability. Target validation studies utilized phenotype restoration in SV40‐HCEC and siRNA function in the human immortalized corneal epithelial cell line, TCEpi. Validation endpoints included multiplex cytokine analysis and high content analysis to understand toxicant effect and target function. Approximately 20 target genes entered in vitro validation studies for each toxicant, but only one gene was in common, NR3C1, a glucocorticoid receptor. For each toxicant, 5‐10 target genes entered testing in mice. These results demonstrated that the drug chlorpheniramine (target HRH1) was effective in preventing central progression of corneal neovascularization in CP‐induced corneal injury during 8 weeks of study even after treatment was withdrawn 2 weeks post‐exposure. In conclusion, these findings show that there is little similarity in the early molecular responses to these three toxicants and suggest that some early interventions for toxicant ocular injury may need to be toxicant‐specific.