bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Brainstem mu‐Opioid Receptors (mORs) Increases Respiratory‐Related Motor Burst Amplitude in Neonatal Rat Brainstem‐Spinal Cord Preparations
Authors: Gumnit Maia G. , Watters Jyoti J. , Baker Tracy L. , Johnson Stephen M.
Published: May 2022

Mu‐opioid receptors (mORs) located on brainstem respiratory neurons are part of an intricate peptidergic neuromodulatory system that regulates breathing. The endogenous role of mORs in respiratory motor control, however, is poorly understood. To explore this question, very low (nanomolar) concentrations of mOR agonist drugs were bath‐applied to neonatal rat (P1‐P3) brainstem‐spinal cord preparations while measuring respiratory‐related motor output from spinal C4‐C5 rootlets containing phrenic motor neuron axons. When DAMGO (synthetic mOR agonist) or endomorphin‐2 (endogenous mOR ligand) were bath‐applied for 15 min at 20‐100 nM, respiratory motor burst amplitude increased dramatically with a modest decrease in burst frequency. Burst amplitude facilitated by 45 ± 38% and 32 ± 24% (mean ± stdev) above baseline for 50 nM DAMGO (n=8) and 100 nM endomorphin‐2 (n=10), respectively (p<0.001). To test whether respiratory motor output on thoracic ventral roots was altered by mOR activation, brainstem‐spinal cord preparations were isolated to include spinal segments C1‐T8. Bath‐applied DAMGO (50 nM) induced burst amplitude facilitation on thoracic roots T4‐T6 and cervical roots C4‐C5 by 62 ± 19% and 38 ± 36%, respectively (p<0.012). To test whether mOR‐induced burst facilitation is due to brainstem or spinal mechanisms, a split‐bath chamber with a barrier at C1‐C2 was used to separately bathe the brainstem and spinal cord compartments. DAMGO (50 nM) application to the spinal cord (C2‐C8) had little effect on respiratory motor output measured from C4‐C5 rootlets, whereas DAMGO application to the brainstem increased C4‐C5 motor output by 34 ± 19% (p<0.004). These data suggest that mOR activation in the brainstem can transform the respiratory network to produce a motor pattern with decreased frequency balanced by increased amplitude, with relatively little change in overall neural output. Given that mOR‐induced facilitation was observed in both cervical and thoracic motor neuron pools, we hypothesize that burst amplitude facilitation is due to mOR activation on rhythm‐generating neurons or premotor neurons in the brainstem. This study reveals that endogenous mOR activation modulates the neural control of breathing in a manner that is potentially different from respiratory depression, which is more commonly associated with opioids.
The Codependent Expression of Yeast RNA Polymerase I Rpa34/Rpa49 Heterodimer Subcomplex
Authors: Baylor Kairo N. , Fakhouri Aula M. , McNamar Rachel , Rothblum Lawrence I. , Knutson Bruce A.
Published: May 2022

Dysregulation of RNA polymerase I (Pol I) is a hallmark of many cancers and several developmental disorders. Pol I is a multi‐subunit complex that synthesizes ribosomal RNA (rRNA), the integral structural and catalytic component of the ribosome. A key yeast Pol I subcomplex that functions in nearly every step of the transcription cycle is the Rpa34/Rpa49 heterodimer. Recent studies with the heterodimer's human counterpart have shown species specific differences in terms of the heterodimer’s importance for cell growth and functional domain requirements. To further explore these differences, we used the yeast model system to show that similarly to humans, the yeast heterodimer subunits exhibit codependent expression. We further analyzed this codependence in terms of specific heterodimer domain requirements and the level of gene expression the codependence occurs. We found that Rpa34/Rpa49 codependence occurs at the post translational level in a manner dependent on Rpa49’s dimerization domain. Overall, our findings suggest that the codependent expression of yeast and human Rpa34/Rpa49 subcomplexes is an evolutionarily conserved feature that may apply to other Pol I, II, and III subcomplexes.
Differential responses of receptor tyrosine kinases C‐Met and EGFR in mice
Authors: Xue Yuhua , Mars Wendy M. , Bowen William , Orr Anne , Stoops John , Michalopoulos George K.
Published: May 2022

In previous studies, we have demonstrated the critical roles of EGFR and HGFR (c‐Met) for liver regeneration and for conversion of hepatocytes to cholangiocytes by a “clean” partial hepatectomy model, in which hepatocytes and cholangiocytes grow rapidly and synchronously. In contrast, the vast majority of liver diseases involve a prolonged pathologic duration, therefore multiple types of cells participate and play different roles in the variant stages. However, it is not fully understood how EGFR and Met play their roles in this complicated setting. In this study, we explore the role of MET and EGFR signaling pathways in biliary injury after common bile duct ligation. Mice with inhibition of EGFR signaling alone (EGFRi) showed abolished hepatocyte proliferation and biliary duct cell proliferation (Ki67 immunochemistry staining) at 2, 5, and 7 days after BDL. However, Met knockout mice (KO) showed increased proliferation of cholangiocytes at 2, 5, and 7 days. Met KO mice subjected to BDL surgery had an increase of the CK19‐ marked ductular reaction at 2, 5, and 7 days. In contrast, EGFRi mice showed a reduced ductular reaction at the same time point. There was enhanced immunohistochemistry for Sox9 in EGFRi mice, but absence of its expression in Met KO mice at 7 days post‐BDL. From western blot results of total liver tissue lysate, we found increased EpCAM expression after BDL at 7 days in Met KO but not in EGFRi mice. In addition, we also observed that both p‐Merlin and cleaved p‐Mst1, components of the Hippo pathway, decreased only in Met knockout mice. Mice with combined disruption of Met and EGFR signaling became very ill by 24 hours and died at 1 to 2 days after bile duct ligation. From Kaplan‐Meier analysis, both EGFR inhibition and Met knockout mice had decreased survival rates starting at 11 days, though the survival rate of Met KO mice was overall much better than EGFRi mice by around 40%. We also observed that p‐Ezrin (Thr567) was downregulated in either Met KO or EGFRi mice, while the total Ezrin expression did not change.

Conclusions

Our study demonstrates that both c‐Met and EGFR control biliary response to bile duct ligation. The cholangiocyte responses, however, were very different between these two receptors. Their combined disruption led to acute liver failure and early death following bile duct ligation. The receptor tyrosine kinases c‐Met and EGFR may play different roles in the biliary injury‐repair responses, including proliferation, trans‐differentiation, and bi‐potential progenitor cells activation.
Conservation of Ilp4, Pdk1 and HDAC4 Across Drosophila Species
Authors: Harris Lindsey , Hark Amy T.
Published: May 2022

The degree of gene conservation is influenced by many factors including relative expression levels and number of interactions of the gene and its protein product. In collaboration with the Genomics Education Project, the conservation of several insulin signaling pathway genes and their protein products within Drosophila has been investigated. Insulin‐like peptide 4 (Ilp4) functions in the insulin signaling pathway by binding to the insulin receptor, thus triggering the phosphatidylinositol 3‐kinase pathway, which involves Phosphoinositide‐dependent kinase 1 (Pdk1). Another player in the insulin‐signaling pathway investigated in this work is Histone deacetylase 4 (HDAC4), which catalyzes the removal of acetyl functional groups from histone proteins, ultimately decreasing the expression of associated genes. Predicting the relative conservation of these genes is not trivial. For example, Ilp4 is at the beginning of the pathway and has one contact according to the Flybase physical interaction network. This might indicate the potential for greater evolutionary change over time, but contradicting this hypothesis is this insulin‐like peptide’s importance as a ligand in the pathway. Pdk1 also shows one contact on Flybase, but is located towards the beginning of the pathway, suggesting a relatively higher evolutionary rate. HDAC4 plays a role in many other cellular processes and so should be well conserved. To evaluate conservation, we first used BLAST to identify orthologs of these three genes across several Drosophilaspecies. We then evaluated conservation in terms of amino acid identity, exon length, number of exons, and degree of synteny using genomic sequences, BLAST, and evidence such as gene prediction tracks and RNA expression data available on the UCSC Genome Browser. Thus far, Pdk1 has demonstrated the greatest similarity over multiple species, with less conservation being observed in Ilp4 and HDAC4. This suggests the complexity in determining the interplay between number of protein contacts and pathway position on evolutionary rate.
A reducible comparison of 2D vs. 3D HepG2 culture‐derived sEV characteristics and cancer pathway‐related miRNA content
Authors: Hood Joshua L. , Schroeder Luke A. , Bardi Gina T.
Published: May 2022

Hepatocellular carcinoma cells (HCCs) produce small extracellular vesicles (sEVs or exosomes) to enable tumor survival, including inactivation of anti‐tumor macrophage immune responses. For sEV studies in vitro, 2D adherent culture cells are typically used to manufacture sEVs. In contrast, 3D matrix‐based culture systems closely simulate in vivo tissues but produce difficult to isolate sEVs. To address this issue, we developed a reducible 3D suspension HCC spheroid culture system to generate easily obtainable sEVs for investigations. The objective was to evaluate biophysical and cancer pathway‐focused miRNA content differences between HCC HepG2 sEVs produced in 2D adherent vs. 3D spheroid suspension culture. To summarize methods, sEVs were isolated from 2D and 3D cell culture using differential ultracentrifugation and size and zeta potential biophysical characteristics were determined by nanoparticle tracking analysis. Fold regulation of 2D and 3D derived sEV miRNAs, compared to their source cells and one another, were determined by qRT‐PCR, and statistically significant differences were determined by Student’s t‐test. Results indicated that HepG2 sEVs generated from 2D adherent or 3D spheroid suspension culture do not differ significantly in terms of size (~130 nm) and zeta potential (< ‐30 mV) characteristic of sEVs. A comparison of 2D vs. 3D sEVs revealed differences in the enrichment of let‐7a‐5p (decreased in HCC in vivo), miR‐21‐5p (enables HCC drug resistance), and miR‐126‐3p (impairs HCC tumor volume in vivo). let‐7a‐5p and miR‐21‐5p were downregulated, while miR‐126‐3p was upregulated in 3D sEVs compared to 2D sEVs (control) respectively. Overall, similarities and differences between 2D and 3D sEV miRNA content were observed relevant to HCC pathogenesis. In conclusion, the 3D HepG2 spheroid suspension model provides an additional reducible level of HCC sEV investigation to augment traditional 2D sEV studies while better simulating an in vivo 3D source of easily obtainable HCC sEVs. Our 3D model could be used in conjunction with typical 2D EV culture systems to streamline the identification of candidate sEV‐based biomarkers and therapeutic targets for HCC and other cancers.
Effects of flow and polycystin‐1 dysfunction on ATP release in the inner medullary collecting duct of the kidney
Authors: van Megen Wouter H. , van Waes Charlotte R. , Peters Dorien J. , van der Wijst Jenny , Hoenderop Joost G.
Published: May 2022

After filtration of blood by the kidney, the remaining pro‐urine flows through the renal tubular system with a flow rate that can vary considerably. These variations in pro‐urinary flow can be sensed by mechanosensing proteins on the epithelial cells lining the renal tubules. An important mechanosensing protein is polycystin‐1 (PC1), which is thought to be a key sensor of fluid flow. Mutations in PC1 give rise to autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid‐filled cysts in the kidney. Previous research has highlighted a role for the ATP release channel pannexin‐1 (PANX1) in the increased flow‐induced ATP release in a Pkd1‐/‐ cell model of the distal convoluted tubule. However, it remains unclear if other purinergic signaling components are also involved, especially in the collecting duct.

Using the mouse inner medullary collecting duct 3 (mIMCD3) cell line in combination with microfluidic experiments, we show here that a similar flow‐induced ATP release is present in Pkd1‐/‐ mIMCD3 cells under low (0.32 mL/min) or high (1.27 mL/min) flow application for 1 minute compared to wildtype (WT) mIMCD3 cells. This is despite a six‐fold increase in the expression of Panx1 observed in Pkd1‐/‐ mIMCD3 cells. Moreover, application of the specific PANX1‐inhibitor BB‐FCF revealed that PANX1 did not drive the high flow‐induced increased ATP release. Accordingly, intermediate flow application (0.47 mL/min) for 3 hours did not increase Panx1 expression in WT mIMCD3 cells compared to the static condition. Flow did not increase the expression of any of the known purinergic receptors in the collecting duct cells compared to the static condition. We did observe a two‐fold increase in gene expression of the putative ATP release channel connexin‐30.3 (CX30.3) compared to static conditions. Moreover, we also report a three‐fold increase in CX30.3 gene expression in Pkd1‐/‐ mIMCD3 cells compared to WT cells.

In conclusion, we show here that flow‐induced purinergic signaling in the collecting duct greatly differs from previously published work on the distal convoluted tubule. Specifically, in the inner medulla of the collecting duct, PANX1 may not be the key flow‐regulated ATP release channel. Instead, CX30.3 may facilitate flow‐induced ATP release in this segment in health and in ADPKD.
Comparative Assessment of the Content of Immunoglobulins in the Blood Serum of Calves Obtained From Healthy Cows and Cows with Genital Mycoplasmosis
Authors: Nikitina Anastasia , Vasiliev Roman , Kovalev Sergey , Trushkin Vyacheslav
Published: May 2022

The genital form of mycoplasmosis is a common disease among cattle in the North‐West region of the Russian Federation. In the initial period of illness, cows retain reproductive function and are capable of maintaining pregnancy. It is known that any mother's illness during pregnancy affects the fetus's body, including its immune system. Based on this, it is interesting to compare the immunoglobulin composition of the blood serum of calves obtained from healthy cows and mothers with genital mycoplasmosis. For the experiment, two groups of calves were formed at the age of 7‐9 days, 8 animals in each. The first group (G1) ‐ calves obtained from cows with a positive PCR test for Mycoplasma spp. and serological identification of M. bovigenitalium, without clinical signs of vaginitis and cervicitis. The second group (G2) ‐ calves from clinically healthy cows with negative PCR test for Mycoplasma spp. Blood was obtained from each group, serum was separated, and the content of immunoglobulins (Ig) of classes G, M, A was determined by discrete precipitation. The study results show that the total content of immunoglobulins in the blood serum in calves from G1 was 9.91 ± 0.27 g / l, and in calves from G2 ‐ 13.8 ± 0.48 g / l, which was 39% lower (P <0.001). As for the classes of immunoglobulins, the level of IgG in calves from G2 was 9.55 ± 0.32 g / l, and in calves from G1, it was almost two times lower ‐ 4.79 ± 0.17 g / l (P <0.001). It was found that the concentration of IgM in the blood serum in calves from G2 was 2.46 ± 0.19 g / l, and in G1, it was 13% lower (P <0.05). The change in the level of Ig A was different, so in calves, from G2, it was 1.79 ± 0.12 g / l, and in calves, from G1, it was significantly higher ‐ 3.24 ± 0.15 g / l (P <0.001). The results of the study show that mycoplasma infection in pregnant cows affects the immunological status of calves. This is manifested by a decrease in the total content of immunoglobulins in the blood serum of calves and a redistribution of their classes: an increase in the level of Ig A and a decrease in the concentration of Ig G and Ig M, relative to calves obtained from healthy cows.
Effect of cadmium on aminotransferases serum activity in European carp
Authors: Krasnovskaia Marina , Karpenko Larisa , Polistovskaia Polina , Bakhta Alesya , Enukashvili Abram , Kozitcyna Anna , Ivanova Katerina
Published: May 2022

In many densely populated areas of the Earth, there is an excess amount of heavy metals in soil, air and water, a significant part of which comes from industrial pollution. So it is important to evaluate possible effects of heavy metals in biological models.

Aim of this study is to asset toxic influence of heavy metals in fish. This research was intended to analyze aminotransferases (ALT, AST) serum activity under the influence of various cadmium concentrations in European carp. Intracellular enzymes activity level elevation in serum is a sign of mass cell destruction and consequently is a sign of pathological process. In this experiment 3 groups of fish (European carp) were formed, 10 fish in each group: control group; 1‐experimental group – fish were contained in water with 0.05 mg/l cadmium acetate concentration (10 times higher than fishery waters permissible exposure limit according to the laws of the Russian Federation); 2‐experimental group – fish were contained in water with 0.5 mg/l cadmium acetate concentration (100 times higher than fishery waters permissible exposure limit according to the laws of the Russian Federation). Exposure time was 4 hours. The blood samplings were taken from heart by syringe and placed in dry test tubes. After samplings centrifugation in non‐hemolysed serum samples ALT and AST activities were assessed. Results presented as mean±standard error of the mean. Student's t‐test was used after proving normal distribution. Level of significance is labeled as p<0.05.

The study revealed that there were meaningful elevation of ALT and AST serum activity (p≤0.05) by 44.22% (30,92±1,42 IU/l) and 15.36% (261,5±8,07 IU/l) accordingly in 1‐experimental group in compare with control group (21.44±0.72 IU/l и 226.68±4.35 IU/l). In case where the cadmium acetate concentration were 100 times higher than fishery waters permissible exposure limit were meaningful elevation of ALT and AST serum activity (p≤0.01) by 88.82% (38,34±0,19 IU/l) and 71.53% (388,82±14,57IU/l) accordingly in 2‐experimental group in compare with control group.

After analyzing the received data there were established meaningful aminotransferases serum activity elevation under the influence of various cadmium concentrations in European carp. This may be due to the activation of gluconeogenesis, which is necessary to maintain an adequate glucose level in conditions of intoxication, which determines the direction of metabolic flows towards the predominance of catabolic reactions. This data is important to be taken into consideration not only during assessing physiological condition in fish, but also to increase understanding of the damage extent in biological models.
Arachidonyl‐2‐ Chlorethylamide alters the Gene Expression of Renin Angiotensin System Components in Primary Cultures of Astrocytes
Authors: O'Connor Ann Tenneil , Clark Michelle A.
Published: May 2022

Objective

To determine the of effect Arachidonyl‐2′‐chloroethylamide (ACEA) on the gene expression of renin angiotensin system (RAS) components in rat astrocytes.

Background

We have previously shown that activation of the Angiotensin type 1 receptor (AT1R) altered the expression of the cannabinoid receptor type 1 (CB1R) in rat astrocytes. Activation of the CB1R can potentially have synergistic or antagonistic effects on AT1R actions. Studies have shown that an overactive RAS contributes to pathological conditions. Thus, we determined the molecular effects of activation of the CB1R by ACEA on RAS components. To investigate this, we determined ACEA and Angiotensin (Ang) II effects alone, then combined, on the mRNA levels of RAS components in astrocytes isolated from the cerebellum of Wistar and Spontaneously hypertensive rats (SHRs). CB1R and RAS components are expressed in the cerebellum.

Methods

Astrocytes were prepared from 2‐3 days rat pups. Quiescent cells were treated with 10nM ACEA and or 100nM Ang II for 12‐24 hours. mRNA levels of RAS components were measured by quantitative polymerase chain reaction.

Results

Ang II predominantly downregulated RAS components in both Wistar and SHR astrocytes at the time points examined. ACEA downregulated AT1R mRNA in Wistar, increased angiotensinogen mRNA in both Wistar and SHR astrocytes, and increased angiotensin converting enzyme mRNA in SHR astrocytes. The effects of combined treatments were similar to Ang II single treatments.

Conclusion

CB1R activation altered the mRNA levels of RAS components but did not appear to have considerable impact on Ang II’s effects on these components. Since mRNA levels might not correlate with protein levels, determination of protein levels and/or activity would complement these findings.
Examining Differences in the Binding Sites of Succinyl Transferases DapD and TabB
Authors: Jimenez Tatiana , Snidman Leah , Calderone Christopher T.
Published: May 2022

TabB and DapD are N‐succinyl transferases that succinylate tetrahydrodipicolinic acid (THDPA) during the biosynthesis of the natural product tabtoxin and lysine, respectively. Although TabB and DapD catalyze identical reactions, and are predicted to form homologous, trimeric left‐handed β‐helical structures, close examination of their sequences reveal that the three‐dimensional arrangements of functionality within their respective active sites diverge significantly. Further examination of the sequences and modeled structures of DapD and TabB suggest that they are representatives of distinct classes of THDPA N‐succinyl transferases, with a small number of amino acids that appear to distinguish “TabB‐class” from “DapD‐class” enzymes. The goal of this work was to test whether it was possible to “swap” one active site for the other within a single three‐dimensional scaffold. In total, we created sixteen DapD and TabB mutants. Four swapped amino acids in DapD for amino acids in TabB and vice versa with the goal of morphing one binding site into the other. The other twelve mutations replaced one amino acid for other similar, yet structurally distinct amino acids. The mutated plasmids were then transformed into E. coli and the corresponding TabB and DapD mutants were overexpressed and isolated. We tested the activities of these mutants via a colorimetric Ellman assay that detected the formation of the reaction coproduct CoASH. Initial results suggest that there is some plasticity within the TabB and DapD active sites, and future efforts are directed to exploring the boundaries of this catalytic flexibility.
Genotoxic Stress Globally Downregulates Transcription in Budding Yeast
Authors: Kaur Pritpal , Nagar Shreya , Mehta Riddhi , Vancura Ales
Published: May 2022

DNA damage response significantly affects transcription. UV exposure of mammalian cells elicits global transcriptional shutdown orchestrated by an essentially enigmatic mechanism. We show here that a brief treatment of budding yeast with hydroxyurea, 4‐nitroquinoline 1‐oxide, or bleocin causes significant reduction in newly synthesized RNAs produced by all three RNA polymerases. This effect is not restricted to S phase and is suppressed by inactivation of checkpoint kinases MEC1 and RAD53, as well as by inactivation of histone chaperones ASF1 and RTT106. In addition, the genotoxic stress leads to removal of TATA‐binding protein from promoters of genes transcribed by all three RNA polymerases and removal of RNA Pol I and RNA Pol II from the corresponding genes. The effect of genotoxic stress on the stability of preinitiation complexes (PICs) does not seem to be significantly suppressed by ASF1, RAD53, or MEC1 inactivation. Together, our data suggest that genotoxic stress affects stability of PICs and production of new RNAs by at least partly independent mechanisms.
Myeloid YAP Inhibition Improves Cardiac Phenotype During Pressure Overload Stress
Authors: Francisco Jamie , Zhang Yu , Nakada Yasuki , Hu Che‐Ming , Zhai Peiyong , Del Re Dominic P.
Published: May 2022

Inflammation is an integral component of cardiovascular disease and is thought to contribute to cardiac dysfunction in ischemic and non‐ischemic models of heart failure. While ischemia‐induced inflammation has been extensively studied in the heart, relatively less is known regarding cardiac inflammation during non‐ischemic stress. Recent work has implicated a role for Yes‐associated protein (YAP), a transcription co‐factor, in modulating cardiac inflammation in response to ischemic injury; however, whether YAP influences inflammation in the heart during non‐ischemic stress is not described. We hypothesized that YAP mediates a pro‐inflammatory response during pressure overload (PO)‐induced non‐ischemic injury, and that targeted YAP inhibition in the myeloid compartment is cardioprotective. In mice, PO elicits an immune response characterized by infiltration of myeloid cells that precedes cardiac dysfunction. Myeloid cells isolated from the heart after 7 days PO showed evidence of increased YAP activity. Myeloid‐specific YAP knockout mice (YAPF/F;LysMCre) were subjected to PO stress. After 4 weeks, cardiac hypertrophy was similar between myeloid YAP KO mice and controls. However, systolic dysfunction, cardiac fibrosis, apoptosis, “fetal gene” induction, and additional indicators of pathological remodeling were attenuated in myeloid YAP KO mice compared to controls. Additionally, inflammatory gene expression and CCR2+ macrophage infiltration in the myocardium were significantly attenuated in myeloid YAP KO mice after PO, indicating reduced inflammation compared to controls. Conversely, angiogenic indicators and pro‐resolving genes were enhanced in myeloid YAP KO hearts. Experiments using primary bone marrow‐derived macrophages (BMDMs) from YAP KO and control mice demonstrated that YAP suppression attenuated “M1‐like” inflammatory gene expression, while “M2‐like” resolving genes were augmented following stimulation. The inflammasome is a multiprotein complex and important facilitator of cytokine processing that mediates inflammation in the injured heart. We observed attenuated inflammasome priming and function in YAP deficient BMDMs, as well as in myeloid YAP KO hearts following PO, indicating disruption of inflammasome induction. Finally, we leveraged nanoparticle‐mediated delivery of the YAP inhibitor verteporfin to test the translational potential of targeting YAP to prevent heart failure. An alternate‐day dosing regimen of drug‐loaded nanoparticles attenuated inflammation and fibrosis, and improved myocardial capillary density after 2 weeks PO compared to DMSO nanoparticle control treatment. Together these data implicate myeloid YAP as an important molecular nodal point that facilitates cardiac inflammation and pathology during PO stress and suggest that selective inhibition of YAP may prove a novel therapeutic target in non‐ischemic heart disease.
Musculoskeletal Differences Between Amputated and Non‐Amputated Lower Limbs
Authors: Finnerty Caitlyn , Finco MG , Ngo Wayne , Holley Bethany , Menegaz Rachel A.
Published: May 2022

People with lower limb amputations frequently experience greater risks of musculoskeletal injury. Forces active during walking help to develop and maintain the shape, volume, and strength of musculoskeletal tissues. Conversely, altered walking patterns following limb loss may lead to atrophy of muscle and bone tissues. Reductions in joint spaces are indicative of excess stress placed on the limb, which may lead to osteoarthritis. Bone loss in high stress regions like the femoral neck can reduce the bone’s ability to resist compressive or rotational movements, making the bone more susceptible to fracture.

The aim of this study was to measure musculoskeletal differences between an individual's residual (amputated) limb and intact (non‐amputated) limb to identify structures vulnerable to injury. We hypothesized that the residual limb, compared to the intact limb, would show: 1) less muscle mass and more fat as indicators of muscle atrophy, 2) wider hip and knee joint spaces as indicators of osteoarthritis in the intact limb, and 3) decreased femoral neck width as an indicator of fracture risk.

CT scans of 10 males (42‐79 years) were obtained from the New Mexico Decedent Image Database. 3D Slicer software was used to measure gross skeletal properties, hip and knee joint dimensions, and cross‐sectional muscle and fat tissue areas at the midshaft. A Wilcoxon Signed‐Rank test was used to assess the differences between residual and intact limbs. The significance level was set at α ≤ 0.10 due to a small sample size. Compared to the intact limb, the residual limb had significantly less muscle tissue area (p=0.010) and a significantly narrower femoral neck width (p=0.077). No significant differences were found in hip or knee joint spaces between limbs.

In agreement with hypotheses 1 and 3, these results suggest residual limbs are at increased risk of muscle atrophy and femoral neck fracture compared to intact limbs. Loading inequalities between the residual and intact limb likely contribute to these results. A better understanding of the structural properties associated with musculoskeletal atrophy could inform targeted therapies to reduce the likelihood of injury in this population. Future studies will assess biomechanical properties, such as moment of inertia, to better understand the residual limb’s ability to withstand torsional forces and fracture. Additional data on how musculoskeletal tissues respond to unloading at multiple structural levels can improve clinical interventions for lower limb strength and function in amputees.
The Gut‐Brain Connection: The Inflammatory Role of IL‐6 Caused by a Western Diet
Authors: Sotelo Alexis A. , Kania Barbie , Ty Darren , Wisco Jonathan
Published: May 2022

Introduction

An increase in chronic proinflammatory immune activity is recognized as an increasingly fundamental element in relation to neurodegenerative disorders. Alzheimer’s disease (AD) is one of the most common neurodegenerative disorders, with recent evidence suggesting the Western Diet as a source for systemic inflammation. The communication fusing the gut to brain axis has been investigated through hepcidin, derived from both hepatocytes and dendritic cells. Hepcidin could potentially play an anti‐inflammatory role in the process of gut dysbiosis through a means of either a localized approach of nutritional immunity, or a systemic approach. Similarly, IL‐6 is a part of the gut‐brain axis. The levels of IL‐6 expression are affected by the gut microbiota and therefore sought to interplay a key role in cognitive function and decline, which could ultimately lead to several neurodegenerative diseases such as Alzheimer’s. The production of IL‐6 is increased by inflammation and associated with sepsis, endotoxemia and treatment with pro inflammatory cytokines. The release of this cytokine peripherally influences the CNS by a ting on the microglia to stimulate a state of neuroinflammation. During inflammation, proinflammatory cytokine IL‐6 is released from the intestinal mucosa and activates the Vagus nerve afferents. Hence, it could conceivably be hypothesized that IL‐6 activates gastrointestinal submucosal neurons through the brain‐gut axis. The aim of this study is to conduct a systematic review of literature through the search of available online medical databases containing information pertaining to gut dysbiosis and the primary communication between the brain‐gut axis.

Methods

A total of 80 articles were retrieved from the databases of Pubmed, Google Scholar and Frontiers. Our method of assessment analyzed prominent keywords in our study pertaining to the small intestines, brain, liver, hepcidin, TMAO, BDNF, iron, astrocytes, WD, microbiota, gut microbiome, gut dysbiosis, iron, inflammation, oxidative stress, IL‐6, and vagus nerve. After reading the full‐text then extracting the findings from each study, our team organized those findings of brain, liver and gut biomolecules in relation to maintaining homeostasis and prevention of inflammation. The databases were assessed fall of 2021 following exportation of articles to a bibliographical database for further screening processes.

Results

The results of this study indicate the optimal state of a healthy brain is IL‐6, hepcidin, ferritin and iron are at baseline levels. Whereas, in a dysbiosis state likely caused by WD, the brain responds by upregulating IL‐6, hepcidin and ferritin levels.

Conclusions

It would be interesting for a future study to experimentally focus on whether hepcidin upregulation induces the downregulation of iron in the neuron, or if the upregulation of iron in the neuron induces the downregulation of hepcidin in the setting of neuroinflammation.
L‐DOPA dioxygenases from diverse natural product pathways
Authors: Klugh Kameron L. , Jones Paige , Yoza Riri , Peterson Larryn W. , Colabroy Keri L.
Published: May 2022

L‐DOPA dioxygenase is part of a mini‐pathway to the synthon 3‐vinyl‐2,3‐pyrroline‐5‐carboxylic acid (VPCA) that is elaborated and embedded within the final product structures of lincomycin, anthramycin, sibiromycin, tomaymycin and hormaomycin. Using the VPCA mini‐pathway as a starting point, we searched sequence space to identify novel natural product pathways containing a VPCA synthon. From among these novel natural product pathways, representative L‐DOPA dioxygenase gene products from Streptomyces hygroscopicus subsp. jinggangensis and Nocardia arthriditis were studied as pure proteins for their stability and activity on L‐DOPA and related catechols in steady‐state assays for catechol cleavage. These results were analyzed in comparison to characterized L‐DOPA dioxygenases from Streptomyces lincolnensis and Streptomyces sclerotialus.
Retinoic acid levels control tooth morphology in fish
Authors: Gibert Yann , Jackman William R.
Published: May 2022

Misshapen teeth are highly common in humans. In most instances, they are due to genetic mutations in genes controlling the morphogenesis of teeth. This is the case for the Runx2 gene, which once mutated leads to cleidocranial dysplasia (CCD) an autosomal dominant genetic syndrome presenting with peg‐like teeth in humans. While many factors have been identified in the process of tooth morphogenesis, currently, little is known about how cell signaling participates in establishing organ shape during odontogenesis. Our previous work has identified the signaling molecule retinoic acid (RA) to be one of the main actors of tooth induction in fish and recent data suggest that RA also plays a role during tooth morphogenesis. Fish and zebrafish, in particular, are good models to study the genetics, cell and molecular biology, and organogenesis of the tooth in vertebrates. The objective of the proposed studies is to understand the roles played by retinoic acid during tooth morphogenesis in different fish species, to develop a fish model of CCD, to clarify the role played by Runx2 during tooth morphogenesis and to understand its link with RA signaling. By exposing fish embryos to exogenous RA and to an RA inhibitor during tooth morphogenesis we have been able to quantify changes in tooth shape and better understand the mechanism of action of RA signaling during tooth morphogenesis in fish. Exogenous RA exposure alters the shape of the first developing tooth in zebrafish making it narrower and more elongated resembling other tooth shape observed in the wild. RA exposure to a fish species, like the goldfish, with shorter and wider tooth makes it also narrower and more elongated making it more like a zebrafish tooth. At the molecular levels, the expression of tooth epithelium and mesenchyme markers dlx2a; dlx3b; pitx2a; runx2b and lhx6 is expanded in RA treated zebrafish embryo. Our data reveal that the levels of RA in different cells of the tooth germ is controlled by the timing and level of expression of the RA degrading enzyme cyp26b1 in a subset of cells of the developing tooth germ. We hypothesize that modifying the onset of cyp26b1 expression in the tooth germ will, therefore, change the level of RA available in the tooth germ, ultimately altering the shape of the tooth. Finally, our work has revealed more about how RA regulates Runx2 and controls tooth morphogenesis in the development of CCD and during evolution. Our project has revealed that fine tuning of RA level in the developing tooth is a crucial player in the vast tooth diversity observed in fish species in the wild.
Innate Immune System Stimulation During Pregnancy Upregulates Thromboxane Synthesis in Rat Maternal Heart
Authors: Tucker Selina M. , Bradshaw Jessica L. , Cushen Spencer C. , Gardner Jennifer J. , Ricci Contessa A. , Goulopoulou Styliani
Published: May 2022

Background

Infections during pregnancy are associated with adverse maternal and fetal outcomes. We previously showed that exposure to immunostimulatory CpG oligonucleotides (ODN2395, synthetic Toll‐like receptor 9 agonist) during pregnancy induces maternal vascular inflammation and enhances vascular tone in pregnant rats. These outcomes were mediated in part by activation of the cyclooxygenase/thromboxane A2 (COX/TxA2) pathway. The objective of this study was to investigate the impact of ODN2395‐induced immune system stimulation on maternal hearts during pregnancy.

Hypotheses

Exposure to TLR9‐mediated immune system activation during pregnancy upregulates the COX/TxA2signaling pathway in maternal cardiac tissues.

Methods

Rats were treated with a synthetic CpG DNA (ODN2395, 1 mg/kg, intraperitoneal injection) or vehicle (saline) in late pregnancy (gestational day (GD) 14, 16, and 18) and euthanized on GD 20 (term 22‐23). Fetoplacental biometrics were recorded after euthanasia and maternal hearts were collected to assess COX‐1 and COX‐2 expression via western blotting and 6‐keto PGF1α (PGI2 metabolic byproduct) and TxB2 (TxA2 metabolic byproduct) production use ELISA.

Results

Maternal heart weights did not differ between groups (ODN2395: 0.26 g/100 g BW vs. Vehicle: 0.23 g/100 g BW, n≥7, p=0.11). Left ventricular tissues from dams treated with ODN2395 released higher concentrations of TxB2compared to cardiac tissues from vehicle‐treated dams (ODN2395: 0.56 ± 0.06 ng/mg total protein vs. Vehicle: 0.31 ± 0.04 ng/mg total protein, n≥5, p=0.0041) but there were no differences in cardiac 6‐keto PGF1α release between groups (p=0.16). COX‐2 expression was lower in left ventricles from ODN2395‐treated rats compared to vehicle‐treated rats (p=0.009). There were no differences in cardiac COX‐1 expression between groups (p=0.27). Exposure to ODN2395 during pregnancy did not affect fetal weights (ODN2395: 2.28 ± 0.03 g vs. Vehicle: 2.25 ± 0.06 g, n≥7, p=0.9) or placenta weights (ODN2395: 0.44 ± 0.02 g vs. Vehicle 0.49 ± 0.01 g, n≥7, p=0.06), but increased fetal‐placental weight ratio (ODN2395: 5.3 ± 0.22 vs. Vehicle: 4.7 ± 0.15, p = 0.04). COX‐2 expression was greater in placental tissues from ODN2395‐treated rats (p=0.004) but there were no differences in placental 6‐keto PGF1α(p=0.51) and TxB2 production (p=0.32).

Conclusion

Exposure to immunostimulatory CpG ODN during pregnancy induces upregulation of TxB2 synthesis in maternal cardiac tissues coupled with a reduction in COX‐2 expression. We suggest that cardiac inflammation during pregnancy may increase maternal risk for future cardiovascular events.
Development of Field‐Based Prediction Equations For Determining Leg Limb Occlusion Pressure
Authors: Neph Sylvia , Plouffe Audrey E. , Vondrasek Joseph E. , Cox Benjamin , Vranish Jennifer E. , Montoye Alexander E.
Published: May 2022

Blood flow restriction and ischemic preconditioning are occlusion techniques used in clinical and athletic populations as rehabilitation techniques and ergogenic aids. Both require determination of limb occlusion pressure (LOP) to know how much pressure to put in a limb‐worn cuff. Measurement of LOP takes sophisticated equipment and designing a low‐cost way of predicting LOP may allow for these occlusion techniques to be more readily used in field‐based settings.

PURPOSE

To determine demographic and other variables that can be used to predict LOP.

METHODS

Participants (n=23, 57% female) aged 18 to 53 years reported to the laboratory for a single session. Participant age, height, weight, body mass index (BMI), body fat percentage, thigh circumference of both legs, systolic, diastolic blood pressure of both arms and mean arterial pressure (MAP), resting heart rate, and single‐leg wall sit times for both legs were assessed. Additionally, participants lay in a supine position and were fitted with an automated system (Delfi) on the proximal thigh, one leg at a time, for determining LOP. LOP was confirmed for each leg using a Doppler ultrasound (GE logique) on the popliteal artery. Then, correlation and stepwise regression analyses were used to determine which variables were significant predictors of LOP.

RESULTS

Right arm MAP (r=0.648) and BMI (r=0.539) had the highest correlations with LOP. Stepwise regression yielded two potential predictive equations to predict LOP. Equation 1: LOP = 2.117*MAP ‐ 6.678 (r2=0.42; standard error of estimate (SEE)=17.6 mmHg); Equation 2: LOP = 1.892*MAP + 2.441*BMI ‐ 52.059 (r2=0.62; SEE=14.6 mmHg).

CONCLUSION

Arm blood pressure was most closely associated with leg LOP, and MAP along with BMI might serve as useful measures for predicting LOP in field settings. Cross‐validation is needed to confirm these preliminary findings, and further analysis should explore other variables that may allow for more accurate prediction of LOP in field settings.
Dynamic Muscle Fatigue and Potentiation in Human Plantar Flexors
Authors: Zero Alexander M. , Paris Michael T. , Rice Charles L.
Published: May 2022

Activation history can enhance or impair subsequent contractile responses. Following a brief high‐intensity voluntary contraction, the contractile response for the same submaximal input stimulus will be enhanced (post‐activation potentiation; PAP). However, prolonged voluntary activation can impair contractile responses due to neuromuscular fatigue. Because both effects are dependent on immediate prior activation, a co‐existence between these two competing processes is observed. Specifically, submaximal frequencies are enhanced (PAP) while maximal frequencies are simultaneously depressed (fatigue). Due to preferential enhancement of low frequency activation, PAP can mitigate the effects of prolonged low‐frequency force depression (PLFFD), ‐ a long‐lasting reduction of force evoked at low‐frequencies following fatiguing exercise. Usually, this relationship has been evaluated using isometric contractions. The purpose was to assess the relationship between PAP, muscle fatigue and recovery during and following an isotonic (dynamic) fatiguing protocol through which power (force and velocity) are assessed. It is hypothesized that PAP will attenuate PLFFD during the fatiguing protocol and throughout 30 minutes of recovery.

The plantar flexors of 5 healthy young adult males (n=4) and females (n=1) to date have been tested while lying prone in a Cybex dynamometer. The ankle was at 90° and knee joint at 180° (straight leg). Inelastic large Velcro straps firmly secured the hips, shoulders and thighs to the dynamometer. Participants performed isotonic plantar flexion contractions over a 20° range of motion (90° to 110°) of the ankle joint with a load of 20% of maximal voluntary isometric contraction (MVC) until peak power was reduced by 20%, 50% and 75% of baseline values. At these intervals of power loss, electrical stimulation of the tibial nerve in the popliteal fossa was done to record unloaded power responses at 10 Hz and 50 Hz. Following task failure (75% power loss), recovery was assessed immediately and at 3, 5, 10, 20 and 30 minutes. For these, voluntary contractions (10 concentric at 20% MVC) were done to induce PAP. Evoked unloaded dynamic measurements were done prior to and following the 10 concentric contractions to assess any effect of PAP.

PLFFD is a preferential loss of low frequency force assessed by comparing a stimulated force ratio between responses at 10 Hz to 50 Hz. During dynamic contractions we used a peak power 10:50 Hz ratio to incorporate the factor of velocity. During fatigue the peak power 10:50 Hz ratio was enhanced by ~35% at 20% power loss, by ~15% at 50% power loss, and minimally enhanced (~9%) at task failure (75% power loss). During recovery this ratio was enhanced follow the potentiating contractions compared to non‐potentiated; task failure (~3%), 3 min (~26%), 5 min (~45%), 10 min (~20%), 20 min (~25%) and 30 min (~38%).

These results indicate that during dynamic muscle fatigue and recovery, PAP may mitigate PLFFD to varying degrees.
Deficient regulation of the epithelial Na+ channel (ENaC) activity and impaired urinary conservation of Na+ in Epac1 ‐/‐ and Epac2 ‐/‐ mice
Authors: Zaika Oleg , Tomilin Viktor , Pyrshev Kyrylo , Stavniichuk Anna , Mei Fang C. , Cheng Xiaodong , Pochynyuk Oleh
Published: May 2022

Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1‐/‐ and Epac2‐/‐ mice exhibit a delayed anti‐natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split‐opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the knockout mice fed with Na+ deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker, ESI‐05 and Epac1&2 blocker, ESI‐09 decreased ENaC activity in EpacWT mice kept on Na+ deficient diet but not on the regular diet. ESI‐09 injections led to natriuresis in EpacWT mice on Na+ deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate non‐redundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC over‐activation.