bluuebunny/crossref_metadata_2025_split huggingface dataset:
Directed evolution of single chain antibody library targeting a disintegrin and metalloprotease‐17 (ADAM‐17)
Authors: Raeeszadeh‐Sarmazdeh Maryam , Do Linh 
Published: May 2022

Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) are zinc‐dependent metalloproteinases within the metalloproteinase (MP) super family, which play critical roles in remodeling and degrading the extracellular matrix (ECM). Particularly, ADAM‐17’s enzymatic function in the activation or inhibition of cytokines, growth factors and their receptors, and adhesion molecules via cleavage of ectodomains has made this protease a critical target in various inflammatory diseases and cancers. Small molecule therapeutics targeting MPs have caused severe side effects in patients, due to nonspecific binding towards MPs. More specific and selective protein‐based therapeutics, such as antibodies, offer a promising alternative to small molecule therapeutics. Developing a more selective antibody via direct evolution can be labor‐ and time‐intensive. Thus, an algorithm‐based approach, like machine learning, can be utilized to increase mutational analysis, and create a deeper understanding of the correlation between protein sequence and function.Here, a synthetic single chain antibody (scFv) library that was previously engineered to reduce non‐specific binding, was further engineered via directed evolution, yeast surface display (YSD), and fluorescent‐activated cell sorting (FACS), for improved binding affinity and selectivity to the ADAM‐17 catalytic domain. DNA sequence analysis from each round of FACS indicated that frequent antibody mutations within light and heavy chain complementarity‐determining region 3 (CDR‐L3/CDR‐H3), were responsible for improved binding to ADAM‐17. Improved scFv clones also demonstrated decreased binding affinity towards other MPs. Machine learning was implemented in mutational analysis to further analyze the relationship between sequence and function. The knowledge and tools developed in this research can be used to engineer and design selective enzyme inhibitors. This work will ultimately shed light onto protein sequence‐structure‐function relations using a combination of rational and combinatorial protein design.
Small extracellular vesicles induced gene expression changes in microglia
Authors: Luo Xuan , Jean‐Toussaint Renée , Sacan Ahmet , Ajit Seena 
Published: May 2022

BackgroundSmall 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.MethodssEVs 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.ResultsWe 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.ConclusionsOur 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.
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).ResultsAt 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.
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

ObjectiveCardiovascular 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.HypothesisWe 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.MethodsAs 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.ResultsThe 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.ConclusionsRougher 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.
The Identification of NIFK’s RNA Binding Targets and Its Prognostic Values in Lung Cancer
Authors: Lin Tsung‐Chieh 
Published: May 2022

BackgroundWe 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.MethodNIFK 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.ResultsWe 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.ConclusionWe 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.
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

BackgroundEvery 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.MethodsWe 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.ResultsWe 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.ConclusionBased 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.
Altered Adipose Tissue Inflammatory Markers in Mothers With Gestational Diabetes
Authors: Maloney Alan , Barnas Jillian , Vieira‐Potter Victoria , Kanaley Jill 
Published: May 2022

INTRODUCTIONAdipose 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.HYPOTHESISMothers with GDM will have impaired AT mitochondrial function and increased markers of inflammation.METHODSAT 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.RESULTSIn 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.CONCLUSIONData 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

BackgroundMetastatic 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 ResultsWe 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.ConclusionOur 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.
To each their own: Disparate abilities of Nrf2 activators to protect against various electrophilic and oxidative insults
Authors: Biesterveld Laura M. , Eggler Aimee L. 
Published: May 2022

Electrophilic and oxidative stress contribute to major diseases such as cancer and diabetes. Small‐molecule activators of the Nrf2 cytoprotective pathway show significant promise for prevention and amelioration of chronic diseases by combating these stressors through upregulation of detoxification and antioxidant enzymes. An understudied area is whether distinct small molecular Nrf2 activators can protect cells from all electrophilic and oxidative stressors, or whether certain molecules are better suited to protect against a particular type of stress.In this study, human keratinocyte HaCaT cells were preincubated for 24 h with one of four Nrf2 activators before different stressors were added for an additional 24 h and cell death was assessed. Three electrophiles were tested: sulforaphane (SFN), the subject of over 70 clinical trials, the multiple sclerosis drug Tecfidera (dimethyl fumarate, DMF), and bardoxolone methyl (BARD), in clinical trials for treatment of chronic kidney diseases and other conditions including COVID‐19. SFN, DMF, and BARD target C151 of the Keap1 protein, a negative regulator of Nrf2. Also tested was KI696, an inhibitor of the Keap1‐Nrf2 interaction. Three chemically distinct stressors—hydrogen peroxide, menadione (a quinone that rapidly generates ROS through one‐electron redox cycling), or chlorambucil (an alkylating chemotherapy agent)—were tested. Stressors were added to the cells at their LC50 concentration after pretreatment with a range of cytoprotective compound concentrations. Unexpectedly, SFN pretreatment showed little to no rescue for all stressors. However, DMF rescued the viability of cells stressed with hydrogen peroxide, menadione, and chlorambucil. BARD showed no rescue for hydrogen peroxide‐treated cells. KI696 improved the cell’s ability to survive chlorambucil. The ability of a given Nrf2 activator (or lack thereof) to protect against a given stressor points to a need to better understand the cytoprotective environment (specific enzymes upregulated, glutathione levels, etc.) that best protect against specific stressors.