bluuebunny/crossref_metadata_2025 huggingface dataset:
Weight Gain, Fibroblast Growth Factor‐23, and Vascular Function in Liver Transplant Recipients
Authors: Martin Linsey F. , Chavez Domenico A. , Bohmke Natalie J. , Miller Austin , Bhati Chandra , Wolver Susan , Siddiqui Mohammad S. , Kirkman Danielle L.
Published: May 2022

INTRODUCTION

Liver transplant (LT) recipients experience significant weight gain and an increased risk of cardiovascular disease (CVD) after transplant. The pathophysiology of this cardiometabolic burden is not fully understood. In the general population, weight gain and increased adiposity are associated with elevated circulating levels of fibroblast growth factor‐23 (FGF‐23). Increased FGF‐23 has been linked with CVD in other disease populations. This study tested the working hypothesis that weight gain after LT is associated withincreased levels of FGF‐23, that subsequently contribute to vascular dysfunction, an established precursor of CVD.

METHODS

We enrolled 21 LT patients (M/F: 9/12; Mean ± SEM, Age: 55 ± 2 yrs) and 8 age matched healthy controls (HC; M/F: 2/6; 47 ± 6 yrs). Vascular endothelial function was assessed with flow‐mediated dilation (FMD) of the brachial artery using duplex ultrasound. Aortic pressure waves were synthesized from radial artery waveforms acquired by applanation tonometry and the use of a generalized transfer function. Serum levels of FGF‐23 were assessed by enzyme linked immunosorbent assay (ELISA).

RESULTS

Higher body mass index (BMI) values were reported in LT patients (37 ± 1 kg/m2) compared to HC (26 ± 1 kg/m2, p = <0.001). Weight gain (BMI) was positively associated with serum FGF‐23 values (r = 0.5, p = 0.01). Central systolic pressures (125 ± 3 vs. 110 ± 6 mmHg, p = 0.01) and central pulse pressures (51 ± 4 vs. 39 ± 4 mmHg, p = 0.05) were higher in LT patients compared with controls. There was no difference in endothelial function (FMD) in LT patients (4.59 ± 0.55%) compared to their HC counterparts (6.08 ± 0.48%, p = 0.12). FGF‐23 values were not associated with measures of vascular function.

CONCLUSION

Weight gain is evident in LT patients and is correlated with higher serum FGF‐23 values. However, our findings do not suggest a relationship between serum levels of FGF‐23 and vascular function in LT recipients.
Finasteride induces Epigenetic Modulation of LSP1: A Gene implicated in Neutrophil Actin Dysfunction disease
Authors: Ihentuge Churchill , Csoka Antonei
Published: May 2022

Lymphocyte Specific Protein 1 (LSP1) gene translates F‐actin protein expressed in leucocytes such as neutrophils, lymphocytes, macrophages, and endothelium. It controls neutrophil motility, adhesion to fibrinogen matrix proteins, and transendothelial migration. Overexpression or increased level of Lymphocyte Specific Protein 1 (LSP1) is implicated in a rare immunologic disorder Neutrophil Actin Dysfunction characterized by spontaneous onset of recurrent infections and inflammation of oral cavity, skin, and respiratory tract. In this experiment aimed at studying the methylation profile and genes expression following finasteride treatment, we cultured human Leydig cells in the presence of 0.5 μM finasteride for 14 days and performed whole‐genome DNA methylation analysis using the NimbleGen Human DNA Methylation 3×720K Promoter Plus CpG Island Array and Ingenuity Pathway Analysis. Results identified several genes that were differentially methylated following finasteride treatment. Lymphocyte Specific Protein 1 (LSP1) gene was downregulated through methylation. We therefore deduced that finasteride could be a gateway to the treatment of Neutrophil Actin Dysfunction disease through epigenetic modification and decreased expression of LSP1 gene.
Specificity of Antibodies for the Identification of Annexin 1 (ANXA1) Protein in Various Types of Cancer
Authors: Olascuaga‐Castillo Karyn , Rubio‐Guevara Susana , Cáceres‐Andonaire Elena , Altamirano‐Sarmiento Dan , Mantilla‐Rodríguez Elena , Hilario‐Vargas Julio , Morgado‐Díaz José , Berezovski Maxim
Published: May 2022

Annexins are a well‐characterized multigene family of phospholipid‐binding and membrane‐bound proteins that are Ca2+‐regulated. ANXA1 expression is variable in tumor cells, ranging from high levels to none, which is why it is helpful to know the staining potential of various antibodies against the protein. Our research aims to evaluate the specificity of five antibodies used to detect ANXA1 expression in selecting different types of cancer and normal tissue and the differential specificity between the two groups. We examined a data set composed of 2177 samples from The Human Protein Atlas (HPA) as mentioned, cancer patients:1904 samples, and healthy persons: 273 samples. We examined the specificity of five different antibody types of detecting ANXA1 expression (HPA011271, HPA011272, CAB013023, CAB035987, and CAB058693). The specificity of each antibody against ANXA1 protein was evaluated using a staining scale of 0 ‐ 3.00, where 0 indicates no staining, 0.01‐1.0 low staining, 1.01 ‐ 2.0 medium staining, and 2.01 ‐ 3.00 high staining.

Results

CAB013023 had the highest staining values for both cancer and healthy samples; thyroid cancer and endometrial cancer had the highest staining values (2.75 and 2.80, respectively). Breast, head and neck, and bladder normal tissues stained the most intensely in healthy samples (3.00 in all three cases). The antibody's specificity for identifying ANXA1 expression suggests that this may be used as a prognosis and treatment marker in cancer.
Myeloid cell‐specific β2AR deletion enhances cardiac hypertrophy and alters leukocyte behavior following pressure overload
Authors: Xu Heli , Raja Jhansi , Roy Rajika , Tilley Douglas G.
Published: May 2022

As part of the sympathetic nervous system, immune cell‐expressed β2 adrenergic receptor (β2AR) plays an important role in regulating their responses to cardiac injury. We previously demonstrated that deletion of β2AR in all cells of hematopoietic origin resulted in decreased immune cell recruitment, cardiac repair and survival following acute myocardial infarction, however the impact of myeloid cell‐specific β2AR on chronic heart failure (HF)‐induced remodeling remain unknown. Thus, we examined the impact of myeloid cell‐specific β2AR deletion (Lyz2‐Cre x Adrb2fl/fl (LB2) versus Adrb2fl/fl (b2fl) mice) on transaortic constriction (TAC)‐induced HF. Despite a similar initial decline in ejection fraction (EF) at 4 weeks post‐TAC, LB2 mice displayed a better‐preserved EF versus b2fl mice at 8 weeks post‐TAC, which reached a comparable level by 16 weeks post‐TAC. The temporary improvement of cardiac function in the early stage following TAC may be related to an enhanced compensatory hypertrophy in LB2 mice. Indeed, left ventricle posterior wall thickness was higher in LB2 mice especially in the early stage after TAC (4‐8 weeks). This association with enhanced hypertrophy was also observed in an independent model of angiotensin II‐infused LB2 versus b2fl mice with higher expression of Nppa and Nppb observed in LB2 hearts. Via flow cytometry, we observed lower levels of total bone marrow‐derived leukocytes, inflammatory monocytes, macrophages and neutrophils in the hearts of LB2 versus b2fl mice at 16 weeks post‐TAC, whereas total myeloid cells seemed similar between the genotypes. Notably, among the macrophages present in the heart, there was a higher percentage of anti‐inflammatory CD206+ (reparative) macrophages in LB2 mice. In conclusion, these results show that myeloid cell‐specific β2AR regulates cardiac remodeling during the development of chronic HF, which may occur via modulation of the expansion and phenotypic modulation of distinct myeloid cell populations.
Genomic Annotation of Chromosome 3L in Drosophilaananassae
Authors: Cavanaugh Jayda , Merkhofer Evan
Published: May 2022

The Genomics Education Partnership (GEP) is a nationwide community of more than 100 colleges and universities dedicated to facilitating undergraduate biology education and research. GEP provides training, resources, and mentorship to provide students with experiential learning and research experiences in genomics and bioinformatics. Comparative genomics is important for understanding organismal evolution, adaptation and gene conservation. Comparative genomics also advances the understanding of gene function and homology between species.

Drosophila melanogaster is one of the most commonly used model organisms in biomedical science to study fundamental genetics as well as tissue and organ development. The smallest chromosome in Drosophila melanogaster is chromosome 4, also known as the Muller F element, with an estimated size of 5.2 Mb. While the F element has maintained a similar size in many other Drosophila species, it is substantially larger in at least four Drosophila species, one of which is Drosophila ananassae. The D. ananassae Muller F element is more than 18.7 Mb in size. A portion of the D. ananassae genome was annotated for comparison to Drosophila melanogaster. Annotations were completed to generate gene models for genes in Contig 26 of the D. ananassae 3L chromosome (Muller D element) as a comparison to the Muller F element in this species. This was completed using the UCSC Genome Browser, Gene Record Finder, FlyBase, the Basic Local Alignment Search Tool (BLAST) and gene prediction software, such as GENSCAN.

Models for four genes were generated from this genomic region: CG8757, Hml, Tsp68C and CG3795. These genes showed a high level of conservation when comparing Drosophila ananassae and Drosophila melanogasterorthologues. The Hml gene codes for hemolectin, which has an orthologue in humans known as the vWB factor. The CG3795 gene has orthologs in multiple species, including Drosophila melanogaster and Drosophila bipectinata, and was recently reported to be involved in proteolysis and serine‐type endopeptidase activity. CG8757, which is predicted to have dehydrogenase activity in D. melanogaster, exhibited synteny with the orthologous gene on the 3L chromosome in Drosophila melanogaster. However, the order of these in D. melanogaster was determined to be different than those annotated in D. ananassae, suggesting a genome rearrangement has occurred in this region. Interestingly, homology with another gene, CG9150, was observed as well. Synteny analysis indicated this gene is located on the X‐chromosome in D. melanogaster, and further analysis suggests a genome rearrangement in this region as well. This annotation has provided some insight to potential genome rearrangements that have occurred in the evolutionary history of these species. Further analysis will provide a greater understanding of how Drosophila genomes have changed over time.
Effects of Co‐Use of Nicotine on Cocaine‐Food Choice in Socially Housed Female and Male Cynomolgus Monkeys
Authors: Nader Michael , Johnson Bernard , Coller Michael , Odom Jillian
Published: May 2022

Cocaine use disorder (CUD) continues to be a major public health problem worldwide with 2.0 million Americans reporting current cocaine use. At present there are no FDA‐approved pharmacotherapies for CUD. An important consideration in evaluating mechanisms of CUD and potential treatment strategies is the observation that most individuals suffering from CUD also use tobacco products. However, most preclinical studies investigating cocaine do not include co‐exposure with nicotine. For these studies, socially housed female and male monkeys (N= 5/sex) self‐administered cocaine (saline, 0.001‐0.1 mg/kg/inj) under a concurrent schedule with food (1.0‐g banana‐flavored food pellets) as the alternative reinforcer during daily 1‐hr sessions. Under these conditions, the frequency of cocaine choice increases in a dose‐dependent manner; there were individual‐subject differences in sensitivity, but when nicotine was added to the cocaine solution, we individually chose the highest cocaine dose which resulted in <20% cocaine choice for each monkey. When increasing doses of nicotine (0.01‐0.056 mg/kg/inj) were added to the cocaine solution, non‐reinforcing doses of cocaine were chosen (i.e., >80% drug choice) in 8 of the 10 monkeys; importantly, in no cases were these nicotine doses chosen over food. These data suggest that when a non‐reinforcing dose of nicotine is added to a non‐reinforcing dose of cocaine, drug choice increases to the point that the combination is chosen over the non‐drug alternative. Next, environmental manipulations were studied to determine whether the cocaine+nicotine combination was more resistant to changes than cocaine alone. For these studies, we used a delay discounting procedure similar to that described by Woolverton and colleagues (Huskinson et al., 2015, 2016). We compared the indifference point (IP; 50% choice between cocaine and food) when a preferred dose of cocaine was available vs. food and cocaine was delayed, to the IP value when a lower cocaine dose + nicotine was available vs. food. We found that in each case, the IP value was greater for the cocaine+nicotine combination compared with the cocaine alone choice. These findings suggest (1) that co‐abuse of nicotine and cocaine can potentiate the reinforcing effects of cocaine and (2) that manipulations designed to decrease cocaine choice are less effective under conditions in which co‐use of nicotine is studied. These preliminary studies did not reveal sex differences or effects of social rank. At present, it is not known how nicotine and cocaine interact. One hypothesis is that both drugs together produce entirely novel neurobiological effects compared with either one alone. The behavioral endpoints may look additive, as shown in our preliminary data, but the mechanisms are probably not simply additive. These studies may lead to a more personalized medicine approach to treating CUD.
Measurement of UHRF1 and UHRF2 Interaction With Nucleosomes
Authors: Kostoff Matthew G. , Albaugh Brittany
Published: May 2022

UHRF1 and UHRF2 proteins are multi‐domain epigenetic proteins. These two proteins share many similarities in sequence and structure. Previous research in our lab has shown that these proteins bind to H3K9me3 tail peptides. The purpose of our study is to measure UHRF1 and UHRF2 protein interactions with nucleosomes, which are more biologically relevant binding partners. These experiments will be conducted by electrophoretic mobility‐shift or nucleosomes pull‐down assays. The procedures will be optimized using H3K9me3 and unmodified nucleosomes, with the goal of using the assay to identify novel binding partners for UHRF1 and UHRF2.
Morphological Description of Olfactory Organ Changes after Acute Nitrite Exposure in Xiphophorus couchianus
Authors: Hughes Hannah , Huertas Mar
Published: May 2022

This study will explore the effects of acute nitrite exposure in the olfactory organ of Xiphophorus couchianusthrough histological studies. Exposure to aquatic nitrite is known to affect cardiovascular functionality, respiration, and the endocrine and nervous systems in vertebrates. Fish have proven to be beneficial models for studying the effects of aquatic nitrites on vertebrate models. Specifically, the direct interaction of the olfactory system with the aquatic environment offers the opportunity to explore the direct effect of nitrites on the vertebrate nervous system. The olfactory system in fish is incredibly sensitive and allows them the ability to detect odorants across long distances. Thus, the sense of smell in fish is used in a variety of physiological functions (foraging, migration, reproduction, etc.) critical for the survival of the species. For this study, we propose Xiphophorus couchianus as the model species, a live‐bearing fish that uses chemical communication to coordinate reproductive events. We predict that there will be morphological and functional changes in the olfactory tract of X. couchianus due to acute nitrite exposure.

To assess the effects of nitrite on olfaction, male X. couchianus were exposed to sublethal levels (0.005 mg/L) of nitrite for forty‐eight hours. The control group consisted of several male X. couchianus that were not previously exposed to nitrite. After forty‐eight hours, their heads were sampled and fixed in bouin. The heads were oriented in paraffin blocks and a microtome was used to cut their noses into 7µm cross sections. The olfactory tract cross sections of the exposed fish were then stained with alcian blue and Gallegos Trichrome staining, and then compared to the olfactory tract of the control group. We found that exposure to nitrite in olfactory tissues changes the morphology of the olfactory organ. More specifically, olfactory epithelium showed signs of inflammation and changes in the morphology of olfactory cells (i.e., hypertrophy and hyperplasia of mucus, several types of receptor cells and lamina propia).

To assess the effects of nitrite on olfactory organ function, behavior tests were conducted for both the control and treatment groups. Fish were placed in a two‐choice maze where a food odorant was presented. Fish who were exposed to nitrite showed no preference, whereas the control group clearly preferred the side with the food odorant.

Based on the results of this study it can be concluded that nitrite exposure has adverse effects on the morphology of the olfactory tract in X. couchianus and, consequently, hinders typical functioning of relevant olfactory components.
Catecholaminergic RVLM C1 neurons mediates heart failure decompensation and mortality during salt loading
Authors: Schwarz Karla G. , Toledo Camilo , Díaz‐Jara Esteban , Pereyra Katherin , Del Rio Rodrigo
Published: May 2022

Heart failure (HF) is a complex clinical syndrome affecting over 37 million people worldwide. Although advances in medical therapy have significantly improve HF condition, morbidity and mortality rate are still high and global prevalence is continuously rising. Common clinical features of HF include cardiac sympathoexcitation and disordered breathing, which are strongly associated with poor survival of HF patients. Importantly, we have previously shown that brainstem pre‐sympathetic neurons (C1) from the rostral ventrolateral medulla (RVLM), play a key role in sympathetic regulation in HF. However, the role played by RVLM‐C1 neurons during heart failure decompensation is totally unknown. Accordingly, we aimed to determine the contribution of RVLM‐C1 neurons on survival during HF decompensation. Adult male Sprague‐Dawley rats underwent volume overload induce non‐ischemic HF. After two weeks, anti‐dopamine β‐hydroxylase‐saporin toxin (DβH‐SAP: 2.5ng/150nl; or saline) was bilaterally injected into RVLM to ablate C1 neurons. Animals were then expose to high salt diet (3% Na+ in food and 2% Na+ in water) for 3 weeks to induce transition from compensated to decompensated HF state. At 6 weeks post‐HF induction, echocardiography, cardiac autonomic function, breathing function, blood pressure, and mortality were assessed. Compared to normal chow fed HF animals, HF+Na+ rats displayed an increased cardiac index (15.1 ± 0.6 vs. 18.5 ± 1.3 ml/min/100cm2, HF vs. HF+Na+, respectively) and cardiac sympathetic tone (∆HR to propranolol: 8.7 ± 3.1 vs. 23.4 ± 2.5 bpm, HF vs. HF+Na+, respectively). Furthermore, HF+Na+ animals showed a further increase in the incidence of breathing disorders compared to HF (apnea‐hypopnea index: 8.1 ± 0.6 vs. 12.5 ± 2.1 events/hr, HF vs. HF+Na+, respectively). Lastly, salt loading results in marked decompensation in HF rats being the outcome a significant increase in mortality risk (survival: 100% vs. 10% HF vs. HF+Na+, respectively). We found that partial ablation of RVLM C1 neurons in HF+Na+ prevented further deterioration in cardiac autonomic function and breathing disorders and markedly improved survival in HF rats that underwent salt loading (survival: 10% vs. 75% HF+Na+ vs. HF+DβH‐SAP+Na+, respectively). Our results strongly support the role for RVLM C1 neurons in HF decompensation during salt loading and suggest that RVLM C1 may act as a key nodal point mediating salt induced cardiorespiratory distress, decompensation and mortality in HF.
LncRNA Xist Participates in Signaling Pathways Related to Pulmonary Arterial Hypertension and its Comorbidities
Authors: Predescu Dan , Qin Shanshan , Geraci Mark , Mokhlesi Babak , Predescu Sanda
Published: May 2022

Population‐based studies indicate a female predominance in pulmonary arterial hypertension (PAH) of around 2 – 4 over men for all races/ethnicities and across all ages that have been studied to date. While female gender has long been established as the major clinical risk factor for PAH, other factors such as infections, autoimmune diseases, inflammation, obesity, obstructive sleep apnea and genetic predisposition have also been associated with PAH. Obstructive sleep apnea, a common comorbidity of PAH, leads to intermittent periods of apnea, significant hypoxemia, but importantly, also to hypercapnia both of which, in turn, serve to induce pulmonary arteriolar vasoconstriction and PAH. The mechanistic links between hypoxemia/hypercapnia and vascular remodeling remain unclear. Our studies sought to investigate whether obstructive sleep apnea, contributes to the severity of pulmonary artery endothelial cells (PAECs) PAH phenotype.

We recently demonstrated that the increased expression and activity of the long‐noncoding (lnc)RNA‐Xist, essential for X‐chromosome inactivation and dosage compensation of X‐linked genes, drives the imbalance sex/ratio of idiopathic PAH. Using human male and female PAECs of non‐disease controls and PAH patients, in conjunction with molecular cell biology/ biochemistry, and functional approaches, we have found that the PAECs of a female specimen with PAH and obstructive sleep‐apnea displayed the highest increase in Xist expression and proliferation rate. The augmented Xist expression leads to increased expression of the X‐linked gene Elk1, a regulator of cell cycle progression in a tissue‐ and cell‐specific manner, and repression of Klf2, a key transcription factor for endothelial homeostasis and quiescence. These molecular events, more prominent in PAECs of female with PAH and comorbid obstructive sleep apnea, may account for their hyper‐proliferative phenotype. Klf2 also targets several endothelial signature genes that regulate blood pressure, angiogenesis, vascularization etc. Our studies suggest that lncRNA Xist and its signaling network participate in various pathways related to PAH and its comorbidities.
Oxalate Induced Mitochondrial and Lysosomal Dysfunction in Myeloid Cells is Mediated by IL‐10 Signaling
Authors: Mitchell Tanecia , Kumar Parveen
Published: May 2022

Oxalate is a small molecule found in several foods and is excreted in the urine. Accumulation of oxalate in the kidney can lead to crystal and kidney stone formation. Oxalate crystals have been reported to stimulate inflammation and injure renal epithelial cells. However, the effect of CaOx crystals on monocytes and macrophages is not fully‐characterized. Studies from our laboratory have shown that oxalate reduces mitochondrial metabolism and induces oxidative stress in monocytes and macrophages. The purpose of this study was to investigate additional mechanisms of oxalate‐induced changes in myeloid cells. Healthy adults were recruited for these studies and asked to consume a controlled oxalate diet for three days followed by a high oxalate load. Circulating monocytes and plasma were collected from participants before and after the oxalate load. Monocytes were examined using RNA‐sequencing, and plasma cytokine levels were evaluated using ELISA. Human primary macrophages and THP‐1 differentiated macrophages were used to assess mitochondrial quality control and lysosomal function following 24‐hour oxalate exposure using qRT‐PCR, western blotting, and microscopy. Transcriptomics revealed that 1,197 genes were differentially expressed in circulating monocytes following the oxalate load. Genes related to mitochondrial respiration, mitochondrial membrane potential, mitochondrial dysfunction, and IL‐10 signaling (regulator of mitochondrial metabolism) were significantly modified by oxalate. IL‐10 plasma cytokine levels were also significantly reduced. Macrophages exposed to oxalate had an accumulation of dysfunctional mitochondria and reduced lysosomal integrity. The findings from these studies indicate IL‐10 signaling may play a role in regulating mitochondrial quality control and lysosomal function in myeloid cells in response to oxalate. Additional studies investigating the significance of IL‐10 signaling on myeloid cells during crystal and stone formation are warranted.
IL‐25 Supplementation Induces M2 Macrophage Polarization, Reduces Blood Pressure, and Improves Fetal Weight in Placental Ischemic Rats
Authors: Shields Corbin A. , Tardo Geilda A. , Wang Xi , Giachelli Chelsea , Cornelius Denise C.
Published: May 2022

Preeclampsia (PE) is characterized by maternal hypertension, intrauterine growth restriction (IUGR), and increased macrophage activation. Macrophages can be broadly categorized into pro‐inflammatory M1 macrophages (M1s) and anti‐inflammatory M2 macrophages (M2s). The M1:M2 ratio is significantly increased in PE women. However, the precise contribution of increased M1s to PE pathophysiology remains unclear. Using the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia, we tested the hypothesis that supplementation of RUPP rats with IL‐25, a cytokine that drives macrophage polarization to anti‐inflammatory M2s, will increase M2s, decrease mean arterial pressure (MAP), and improve fetal growth. On gestation day (GD) 14, timed pregnant 10‐12 week old Sprague Dawley rats underwent the RUPP or Sham procedure and a mini‐osmotic pump infusing IL‐25 (0.5µg/day) or vehicle was implanted i.p., in both groups. Carotid catheters were placed on GD18. On GD19, MAP was measured, animals were euthanized, and blood and tissues were collected for analysis. Flow cytometry analysis was performed on placental and renal tissues. M2 macrophages (CD3‐/CD68+/CD163+) were markedly reduced in placentas of RUPP rats compared to Sham (0.2% vs. 4.2% of total macrophages) and in kidneys of RUPP rats vs. Sham (0.19% vs. 1.445% total of macrophages). IL‐25 supplementation elevated M2 populations in placental (3.985%) and renal (5.985%) tissues of RUPP. Likewise, IL‐25 treatment increased M2 macrophages in Sham placental (18.56%) and renal (12.99%) tissues. Increased MAP was observed in RUPP rats compared with Sham (123.5 mmHg vs. 106.5 mmHg, respectively) and was reduced in RUPP + IL‐25 (107.5 mmHg). No changes were observed in MAP of Sham rats treated with IL‐25. Fetal weights were reduced in RUPP vs. Sham (2.035g vs. 2.405g, respectively) and were normalized in RUPP after IL‐25 supplementation (2.425g). IL‐25 supplementation had no effect on fetal weight of Sham rats. Additional analyses will include assessment of blood and tissue cytokines and reactive oxygen species, and vascular function in all groups. In conclusion, IL‐25 supplementation increased placental and renal anti‐inflammatory M2 macrophage populations, reduced MAP, and improved fetal weights in RUPP recipients. These data suggest that macrophage activation is a key contributor to PE pathophysiology and may represent a therapeutic target to improve maternal and fetal outcomes in PE.
The Vascular Basis of Takotsubo Syndrome
Authors: Choban Leah , Godel Sarah , Martin Anna , Kolz Christopher , Winnicki Anna , Pucci Thomas , Kegecik Karlina , Shockling Lindsay , Yin Liya , Chilian William , Ohanyan Vahagn , Dong Feng
Published: May 2022

Takotsubo Syndrome (TTS) is an unmet medical need as there is no “standard of care” with an in‐hospital mortality rate equivalent to patients with an acute myocardial infarction. Characteristic of TTS is systolic ballooning of the left ventricular (LV) apex, simultaneous with contraction of the base. The mechanism(s) underlying this disassociation of mechanical activities in the LV is(are) unknown, however, a murine model may provide insights into this clinical conundrum. Previously, we found that mice null for Kv1.5 channels (KO), a model of inadequate coronary metabolic dilation, exhibit TTS (KOTTC) when stressed by transaortic constriction (TAC). Because of the microvascular link to inadequate dilation, we hypothesized that coronary vessels in the base and the apex of the left ventricle have different phenotypes as shown by variations in gene expression. To address this, we dissected small coronary arteries from the LV apex and base of KO control mice (KOC) and following TAC when the mice exhibited profound TTS. Ventricular function (fractional shortening [FS]) was calculated from echocardiographic (Echo) imaging. The Echo measurements revealed FS of 39±3% and 34±6% in LV base and apex, respectively, in KOC (P=NS). In contrast, FS was significantly changed in KOTTC with FS of ‐9±4% in LV apex and 25±11% in the base (P<0.05, with the negative FS representing systolic ballooning). After the Echo measurements, hearts were removed, placed in a chilled dish, and vessels immediately isolated from the LV apex and base (using a dissecting microscope) were placed in Trizol and flash frozen. Total RNA was extracted and used for RNAseq to profile the transcriptomes of small arteries from the apex and base of KOC and KOTTC groups. RNAseq revealed expression of 197 genes significantly changed in the LV apex versus base of KOTTC mice (P<0.05). The 197 genes represented 24 distinct pathways with some of the most significant comprising neuroactive‐receptor ligand interactions, miRs in cancer, and complement and coagulation cascades. The endothelin pathway was significantly upregulated in small arteries of the LV apex compared to base in KOTTC. Moreover, 460 genes were differentially expressed between the apex of KOTTC versus KOC (P<0.05) representing 37 pathways, including miR’s in cancer and cytokine signaling. The changes in miR expression in cancer may also denote changes associated with cell differentiation and apoptosis. These changes support our concept that regional, apex vs base, differences in vascular gene expression may underlie the basis of Takotsubo Syndrome.
Impact of MeCP2 Overexpression on Myelination Defects Found in Pitt‐Hopkins Syndrome Model Mice
Authors: Freitas Geanne A. , Vermudez Sheryl A. D. , Gogliotti Rocco G. , Niswender Colleen M.
Published: May 2022

Introduction

Studies examining neurodevelopmental disorders (NDDs) have emphasized a relationship between Pitt Hopkins syndrome (PTHS), which is caused by mutations in the Transcription Factor 4(TCF4) gene, and myelination defects that suggest novel molecular phenotypes in NDDs (Phan et al., 2020). Known phenotypic overlap between PTHS and Rett syndrome (RTT) suggests that there are possible common signaling pathways between the two NDDs. We have found that increasing expression of Methyl‐CpG‐Binding Protein 2 (MeCP2), the causative protein in most cases of RTT, decreases hyperactivity and corrects impairments in learning and memory in a mouse model of PTHS (Tcf4+/‐).

Aim

The current study sought to understand if the positive effects of MeCP2 overexpression on behavioral phenotypes in the Tcf4+/‐ model were due to a reversal of myelination deficits.

Methods/Results

We generated four distinct animal genotypes (WT, MECP2Tg1/o, Tcf4+/‐, MECP2Tg1/o; Tcf4+/‐) and performed RNA‐sequencing from the hippocampus and striatum followed by quantitative RT‐PCR, revealing that myelin‐associated genes are differentially expressed in MECP2Tg1/o; Tcf4+/‐ mice in both brain regions. However, Western blotting revealed decreased expression for the myelinating oligodendrocytes (OLs) protein Myelin Oligodendrocyte Glycoprotein (MOG) in mice of both the Tcf4+/‐ and MECP2Tg1/o; Tcf4+/‐ genotypes without changes in the OL precursor cell marker Chondroitin Sulfate Proteoglycan (NG2). We are currently quantifying potential changes in OL morphology and number (mature and immature cells), and myelin synthesis via immunohistochemistry of brain sections.

Conclusions

Altogether, our preliminary results indicate that behavioral phenotypic rescue in MECP2Tg1/o; Tcf4+/‐ mice may not result from a correction of myelination deficits characteristic of Tcf4+/‐animals.
Subcellular Protein Expression of Annexin A1 (ANXA1) in Different Types of Cancer
Authors: Olascuaga‐Castillo Karyn , Rubio‐Guevara Susana , Cáceres‐Andonaire Elena , Caballero‐Aquiño Olga , Mantilla‐Rodríguez Elena , Hilario‐Vargas Julio , Morgado‐Díaz José , Berezovski Maxim
Published: May 2022

Annexin A1 (ANXA1) is a Ca2+‐regulated phospholipid‐binding protein involved in several physiological functions. Further research has shown that this protein might have various roles in cancer development and operate at different levels. This is partially related to the placement of ANXA1 in distinct cellular compartments. ANXA1 is maybe nuclear, cytoplasmic, and membrane‐associated. The goal of this study has been to analyze ANXA1 subcellular localization and expression in different types of cancer.

Methods

We examined 1295 data sets available in The Human Protein Atlas (HPA) (HPA). The research examined the ANXA1 expression in tissue from 1130 cancer and 165 normal samples. A visual categorization method based on staining intensity (I), the proportion of stained cells (F) (F). ANXA1 expression levels as a function of subcellular localization.

Q Score (product of E*I) was calculated by the fraction of stained cells (F) and the intensity (I) (I)

Results

In cancer patients, the predominant subcellular localization was cytoplasmic membranous (53.3 %), whereas, in healthy individuals, the principal was cytoplasmic membranous and nuclear (57.0 %). In terms of staining intensity (Q Score), the highest values were found at the cytoplasmic membranous and nuclear levels (Q Score=5.69). In contrast, the highest Q Score values were found in standard samples at the nuclear level (Q Score=6.0).

Conclusion

The subcellular localization of ANXA1 expression may be a helpful diagnostic marker in various cancer types.
G Protein‐Coupled Receptor Kinase 6 (GRK6) Regulation of Insulin Processing and Secretion
Authors: Varney Matthew J. , Steyaert Wouter , Coucke Paul J. , Delanghe Joris R. , Benovic Jeffrey L.
Published: May 2022

We performed exome sequencing in Type 2 Diabetes (T2D) patients belonging to a T2D family containing two affected and five unaffected individuals revealing an autosomal dominant inheritance pattern. A mutation in the GRK6 gene, coding for G protein‐coupled receptor kinase 6, was identified that segregates with the phenotype. GRK6 belongs to a family of serine/threonine kinases that phosphorylate agonist occupied G protein‐coupled receptors effectively modulating downstream signaling pathways including those involved in metabolism. These receptors like the Glucagon‐like peptide‐1 receptor play a critical role in the regulation of insulin processing and secretion and are the target of several approved therapeutics. Therefore, we wanted to determine if GRK6 regulates insulin dynamics potentially contributing to T2D. To start, we performed biochemical assays to unveil the properties of the novel GRK6 mutant. The GRK6 mutant was able to phosphorylate all substrates more effectively than wild type GRK6 but displayed a cytosolic distribution not expected for GRK6. We then utilized pharmacological inhibition and genetic knockdown of GRK6 in MIN6 cells to determine how removal of GRK6 impacts insulin levels after glucose treatment. In MIN6 cells, small molecule inhibition of GRK6 increased insulin secretion but reduced insulin processing. GRK6 knockdown revealed these same processing defects as GRK6 suppression in knockdown cell lines had reduced levels of cellular insulin. Furthermore, these cell lines had attenuated insulin secretion but enhanced proinsulin secretion consistent with decreased processing and the patient phenotype. The altered insulin profile appears to be caused by changes in the proprotein convertases, the enzymes responsible for proinsulin to insulin conversion, as pharmacological inhibition and knockdown resulted in less expression and activity of these enzymes. To support this, we performed rescue experiments where GRK6 re‐expression in knockdown cells restored insulin secretion after glucose treatment. Taken together, our data show that GRK6 regulates insulin processing and secretion in a glucose dependent manner and provides a foundation for the contribution of GRK6 to T2D.
Botulinum toxin injection in masseter muscle evokes musculoskeletal impairment of the masticatory system
Authors: BUVINIC SONJA , Blanco Noelia , Vásquez Walter , Quezada Esteban , Eyquem Andrea , Plotkin Lilian , Kupczik Kornelius , Toro‐Ibacache Viviana , Balanta‐Melo Julián
Published: May 2022

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non‐desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA‐injection in masseter muscle of adult mice.

Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks‐old) by single‐injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral‐ and medial‐pterygoid) and mandibles were dissected after 2‐7‐14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC‐Universidad de Chile (# 20381‐ODO‐UCH). T‐test or Wilcoxon‐test were used for statistical comparisons between BoNTA‐ and Control‐side (n=3‐10, p<0,05).

The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA‐side, as early as 2‐7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time‐dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid’s volumes significantly reduced in the BoNTA‐injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA‐injected side, starting as early as 7d and worsening 14d after the intervention.

In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early‐bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho‐functional monitoring.
Effects of Antiviral Nucleoside Analogues on the Maintenance of the Mitochondrial Genome
Authors: Bisimwa Hyacintha‐ghislaine M. , Ciesielska Elena J. , Kim Noelle , Oliveira Marcos T. , Ciesielski Grzegorz L.
Published: May 2022

The outbreak of the COVID‐19 pandemic prompted the search for effective antivirals. Remdesivir (Gilead Sciences) was the first nucleoside analogue approved by the FDA for COVID‐19 treatment, and currently the FDA considers authorization for the use of another analogue, molnupiravir (Merck). Mitochondrial toxicity, resulting from the interference with mitochondrial DNA (mtDNA) replication, is the most common side effect of nucleoside analogues treatment. In addition, defects to the mtDNA synthesis may give rise to heritable mutations, which may be carried undetected for generations before populating mitochondria to the level that onsets a mitochondrial disease. Therefore, nucleoside analogues need to be thoroughly investigated for their impact on the maintenance and integrity of the mitochondrial genome.

We recently assessed the effect of remdesivir on the maintenance of the mitochondrial genome. We observed that in in vitro DNA synthesis assays, its triphosphate metabolite may impede the synthetic activity of the mitochondrial replicative polymerase, Pol γ, when in excess over nucleotides. Prompted by this finding, we tested the effect of the nucleoside metabolite of remdesivir on the maintenance and integrity of the mitochondrial genome in proliferating fibroblasts. However, we found no evidence for deleterious effects on the integrity of the mitochondrial genome.

We are currently performing an analogical assessment of the effects of molnupiravir on the integrity of the mitochondrial genome. Thus far, we have observed that, unlike remdesivir, molnupiravir is cytotoxic for proliferating fibroblasts and results in a decrease in mtDNA copy number. We are currently testing the impact of molnupiravir on the activity of Pol γ in vitro, and its potential to elicit mutations in the mitochondrial genome in vivo.
Saccharomyces cerevisiae YIL164C/NIT1 is a Putative Nitrilase
Authors: Koyama Erica , Walsh Susan
Published: May 2022

While the yeast Saccharomyces cerevisiae is a well‐studied organism with its genome fully sequenced two decades ago, there remain several hundred uncharacterized open reading frames, ORFans, with no significant sequence homology to other genes. As ORFan roles cannot be determined solely through sequence homology, their functions remain a mystery until both in vitroand in vivobiochemical and cell biology experiments are performed. In collaboration with the Yeast ORFan Gene Project, we used bioinformatics modules to initially characterize one such ORFan, YIL165C.We found that together with the gene immediately preceding it, YIL164C (NIT1), the two genes have significant homology to the nitrilase superfamily of proteins but have low sequence homology to the other two S. cerevisiae nitrilases NIT2 and NIT3. Interestingly, the YIL164C protein alone contains both the conserved catalytic triad and a region that shares homology with prokaryotic nitrilases that specifically target aromatic, rather than aliphatic, nitrile substrates. We additionally experimentally verified that lab strains of S. cerevisiae, such as BY4741, SEY6210, and GA74, have a bonafide stop codon breaking up the two proteins in clones from both genomic DNA and mRNA, while the brewing yeast strain WLP001 (White Labs) and other related yeast strains have a single nucleotide polymorphism (SNP; A to G transition) allowing these two proteins to be expressed as a single, continuous polypeptide chain. To understand the function of both the full‐length YIL164C/YIL165C and the truncated YIL164C protein, we expressed and purified the proteins in bacteria and began initial studies to understand their substrate preference. We hope to identify distinctions in activity, or the lack thereof, between the longer and the truncated proteins. Understanding these enzymes in vitro will provide clues to their in vivo functions and the evolutionary relationships among genes in this superfamily.
MYTHO: A novel regulator of autophagy and skeletal muscle health
Authors: Leduc‐Gaudet Jean‐Philippe , Franco‐Romero Anaïs , Broering Felipe E. , Moamer Alaa , Cefis Marina , Chaffer Tomer J. , Dulac Maude , Marcangeli Vincent , Sedraoui Sami , Shams Anwar , Mayaki Dominique , Huck Laurent , Petrof Basil , Lochmuller Hanns , Sandri Marco , Hussain Sabah N. , Gouspillou Gilles
Published: May 2022

Rationale

The loss of skeletal muscle function can have dramatic health consequence. Skeletal muscle dysfunction are found in a wide range of clinical conditions, including cancer cachexia, starvation, sepsis, and aging. Autophagy is an essential catabolic process that remove damaged cytosolic components. Accumulating evidence indicates that insufficient or excessive autophagy can contribute to the development of skeletal muscle atrophy. We recently identified a novel FoxO‐dependent gene, which we named MYTHO, with the potential to regulate autophagy in skeletal muscles. However, to date, the roles that MYTHO plays in skeletal muscles remain unknown. In this study, we evaluated the functional importance of MYTHO in skeletal muscle structure and function.

Methods

To study the functional importance of MYTHO in skeletal muscle mass and function, we used a combination of genetic strategies in mice to delete or overexpress MYTHO.

Results

MYTHO expression is induced in various muscle wasting conditions, including starvation, denervation, cancer cachexia, and sepsis. Using different genetics approaches, we confirmed that MYTHO is an important regulator of autophagy in skeletal muscle. Importantly, we also found that MYTHO knockdown in skeletal muscles caused various myopathic features, including muscle weakness, accumulation of tubular aggregates, a shift in myofiber type composition and nuclei mispositioning in myofibers. Using animals with muscle specific Atg7 deletion (a model of autophagy inactivation), we demonstrated that the pathological features associated with MYTHO knockdown are independent of its role in regulating autophagy.

Conclusion

We conclude that MYTHO is a central player in regulating skeletal muscle mass and integrity.
Renal Venous Clamping Exacerbates Outer‐Medullary Red Blood Cell Congestion during Ischemia in Male and Female WKY Rats
Authors: Johnson Chloe , McLarnon Sarah , Sun Jingping , O'Connor Paul
Published: May 2022

Despite receiving approximately 20% of cardiac output, the kidneys are highly susceptible to ischemic injury, which is most prominent in the renal outer medulla. Previous studies have suggested that vascular congestion may explain the susceptibility of this region to injury. Further, increased injury has been reported in models where the renal vein is occluded compared to the renal artery. The goal of the current study was to determine whether renal arterial versus venous clamping would alter the severity of red blood cell congestion in the outer medulla. We hypothesized that venous clamping would result in greater vascular congestion than arterial clamping due to continued inflow of arterial blood.

Ischemia reperfusion (IR) surgeries were performed on male and female WKY rats (10‐14 weeks). For each animal, the renal artery for one kidney and renal vein for the other kidney were clamped for a period 15 (n=7 male, 6 female) or 45 (n= 6 male, 5 female) minutes. The kidneys (right or left) which received arterial or venous clamping were rotated for each animal. Following ischemia, the kidneys were excised while the clamps remained on the vessels, without reperfusion. Vascular congestion of the outer medulla was scored blinded on a scale of 0 (0% congestion) to 5 (100% congestion) in trichrome stained kidneys sections.

We found that there was significantly greater congestion of red blood cells in both the vasa recta and outer‐medullary plexus with venous clamping when compared to arterial clamping for both 15 minutes (p=0.0008, p<0.0001, respectively) (Fig. A) and 45 minutes (p=0.0001, p<0.0001, respectively) (Fig. B). There was no significant effect of sex observed on degree of vascular congestion regardless of the location or duration of the clamp.

Our data indicates that red blood cell congestion is exacerbated by renal venous occlusion, which may explain the increased tubular injury that has been observed in this model. Therefore, future studies will investigate injury following recovery from renal artery versus vein clamping. Understanding the cause(s) of vascular congestion is essential for the development of clinically effective treatment options for acute kidney injury. Novel therapeutic approaches that prevent or reverse red blood cell congestion of the renal medulla may be effective in limiting ischemic acute kidney injury.