bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene expression correlation analysis of livers undergoing normothermic machine perfusion identifies interactions between donor demographics and liver function
Authors: Santiago John C. , Raigani Siavash , Ohman Anders , Uygun Korkut , Yeh Heidi , Sanders Jennifer
Published: May 2022

Background

Liver transplantation is currently the only cure for end‐stage liver disease, but the shortage of donor livers severely limits the effectiveness of transplantation. This shortage is made worse by challenges in preserving and assessing livers pre‐transplant that can result in discarding of functional livers. Normothermic machine perfusion (NMP) is a method of liver preservation that aims to provide physiological conditions while allowing real‐time assessment of transplant viability criteria. The aim of this study was to understand the effect of machine perfusion on the transcriptome and to correlate changes in gene expression with functional liver metrics.

Methods

Ten discarded livers (5 steatotic, 5 non‐steatotic), rejected by local transplant centers, were obtained from donation after circulatory death (DCD). The livers underwent 12 hours of NMP with oxygenated red blood cells. Serial tissue and plasma samples were collected at various times during perfusion. Plasma lactate samples were used to categorize liver as meeting viability criteria (viable) or not (nonviable) following NMP. Transcriptome sequencing was performed on serial biopsies taken at 0, 3, and 6hr during perfusion. Weighted gene co‐expression network analysis (WGCNA) was performed to correlate each module with demographics and functional metrics. Pathway and transcription factor analysis were performed on significant modules of interest.

Results

Expression data at 0, 3 and 6 hr of perfusion was used to construct the weighted co‐expression network. Gene co‐expression modules were then correlated with various clinical demographics and functional data collected during NMP. To evaluate the functional association of genes in the modules that significantly correlated with arterial lactate levels, we performed Gene Ontology enrichment analysis. By using lactate clearance as an indicator of transplant viability we were able to identify co‐expression gene sets enriched for biological processes including drug metabolism, immune response, autophagy, vascular development, nutrient metabolism, and gene expression.

Conclusions

Current donor liver assessment models are limited by traditionally accessible parameters and can often lead to discarding potentially viable organs. NMP provides a novel data collection platform that could be used to improve traditional assessment models. By performing WGCNA, we were able to capitalize on an extensive set of metrics to identify interactions between the diverse donor demographics, the functional data collected during NMP and changes in gene expression. This may ultimately allow us to identify novel biomarkers to improve our current methods of donor liver assessment and assist the development of targeted therapeutics for more successful preservation and utilization of diverse donor livers.
TFEB and TFE3 Depletion Increases Cancer Stem Cell Characteristics and EMT in Triple‐Negative Breast Cancer
Authors: Incristi Aurelia , Soleimani Milad , Van Den Berg Carla
Published: May 2022

Breast cancer is currently the second leading cause of death for women worldwide. One subtype of breast cancer with poor prognosis is triple‐negative breast cancer (TNBC), which is characterized by the lack of expression of estrogen, progesterone, and human epidermal growth factor 2 (HER2) receptors. TNBC is difficult to treat due to its drug resistance and aggressiveness. Tumor invasiveness is driven by epithelial‐mesenchymal transition (EMT), which allows cells to acquire mobility to metastasize. EMT is also relevant to maintaining cancer stem cell (CSC) characteristics. Drug resistance is driven by CSCs, which are slow to divide, thus surviving chemotherapies, the standard of treatment. Thus, new treatments that inhibit CSCs and/or EMT in TNBC are highly desirable. TFEB and TFE3, two transcription factors of the MiT‐TFE family that operate as a complex, are involved in regulating lysosome biogenesis and autophagy. Our preliminary data and other studies have shown that TFEB and TFE3 regulate pluripotency and EMT in various in vitro models such as gastric and pancreatic cancer, but this regulation is poorly understood in TNBC. To develop new targeted therapies for TNBC, this study aimed to investigate the effects of depleting TFEB and TFE3 on CSC abundance and EMT. CSC renewal capacity was assessed via mammosphere assays and flow cytometry using CD44 and ALDH1A. The MDA MB 231 TFEBKO cells grew the highest number and largest mammospheres compared to the control, TFE3 knock‐down, and double knock‐out cell lines. TFEBKO and TFEBKOTFE3sh showed increases in CD44 expression and ALDH1A activity. While ALDH1A positivity was 1.16% in controls, TFEBKO and TFEBKOTFE3sh significantly increased the population to 22.6% and 21.9%, respectively. Western blotting and a wound healing assay were used to evaluate EMT marker expression and migration, respectively. Quantification revealed that TFEBKO had a two‐fold increase in protein expression levels of EMT markers Vimentin, Snail, and Slug, while TFE3sh showed higher protein expression levels of Vimentin and Slug. TFEBKOTFE3sh showed higher protein expression levels of Slug. The TFEBKO cell line had the highest rate of migration when compared to the control. In conclusion, the data showed that knockout of TFEB and combined knockout of TFEB and TFE3 resulted in an increase in stem cell character and EMT, which could correlate to increased tumor metastasis and poorer prognosis. These results are promising to investigate how depletion or inhibition of TFEB and TFE3 can affect TNBC tumor cell stem cell character and EMT.
Electrostatic Interaction Between Rotor and Stator Subunits in the Fo Motor of E. coliATP Synthase
Authors: Stewart Ariel D. , Rothrock Mallory M. , Shrestha Rashmi K. , Founds Michael W. , Steed Ryan
Published: May 2022

F1Fo ATP synthase is the ubiquitous biomolecular machine that catalyzes the final step of oxidative phosphorylation and is therefore the primary producer of ATP across all domains of life. The membrane‐embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which provides two aqueous half channels that facilitate proton translocation. The mechanism by which proton translocation is converted into torque on the c‐ring is not fully defined. Previous work has suggested that conserved residues aAsp92, aGlu196, and cArg50 in the proton exit pathway are important for proton transport and possibly for torque generation. To clarify the roles of these residues, we generated 22 mutants and assayed their growth on succinate and in vitro ATP synthesis, H+ pumping, and permeability activities. Mutations of aGlu196 had only a mild effect on proton pumping, while moderately inhibiting ATP synthesis. These results indicate that aGlu196 is likely not interacting with cArg50 but interestingly do suggest that it may have a greater role in ATP synthesis than proton pumping. In contrast, mutations of aAsp92 were not well tolerated, and mutations that reverse the charge of cArg50 caused a substantial defect. These results along with the structural proximity of these residues suggest that they may interact electrostatically across the rotor stator interface.
Constrictor Responses of Cerebral Resistance Arterioles Preserved in Male and Female Rats Exposed to Prenatal Alcohol
Authors: Knecht Tiffany , Saha Partha , Arrick Denise , Watt Michael , Scholl Jamie , Mayhan William
Published: May 2022

Fetal alcohol spectrum disorder (FASD) is the result of exposure to alcohol in utero. This disorder causes lifelong intellectual and behavioral disabilities, as well as cardio and cerebrovascular dysfunction. However, the mechanisms by which these abnormalities arise are poorly understood, and no treatment exists. Past studies have shown that dilation of cerebral arterioles to nitric oxide synthase (NOS)‐dependent agonists is impaired in rats exposed to prenatal alcohol, yet no studies have examined the influence of prenatal alcohol on constrictor responses of cerebral arterioles. We hypothesize that in uteroalcohol exposure may impair the activity of the vascular smooth muscle, which could lead to an alteration in the ability of the cerebral arterioles to constrict in response to endogenous vasoconstrictor agents. Therefore, the goal of this study was to determine whether responses of cerebral arterioles to vasoconstrictors are altered in rats exposed to alcohol in utero. We fed Sprague‐Dawley dams a liquid diet with or without alcohol (3% ethanol) for the duration of their pregnancy (21‐23 days). We then examined the reactivity of the cerebral arterioles of the pups to arginine vasopressin (AVP; 0.1 and 1 nM), thromboxane (U‐46619; 10 and 100 nM), and angiotensin II (1 and 10 µM) in four groups: control male, control female, prenatal alcohol male, and prenatal alcohol female, at two different age ranges: 4‐6 weeks of age and 14‐16 weeks of age. We found that constrictor responses of cerebral arterioles to AVP and the thromboxane analog (U‐46619) were similar in control rats and rats exposed to prenatal alcohol at both age groups for both sexes. In addition, adolescent male and female rats showed no difference to angiotensin II following prenatal exposure to alcohol. However, alcohol‐exposed females exhibited an unexpected dilation to the high concentration of angiotensin II in adulthood, which was absent in males. We suggest that the findings from these studies may have implications regarding the susceptibility of the brain to cerebral ischemic damage. We speculate that impaired vasodilation, with preserved vasoconstriction, can lead to a scenario favoring a decrease in cerebral blood flow during times of increased metabolic demand. This is the first published study to examine the influence of in uteroexposure to alcohol on constrictor responses of cerebral arterioles. Further understanding of how cerebrovascular blood flow may be altered in rats with FASD can ultimately lead to an understanding of how the resulting ischemic damage may relate to intellectual and behavioral abnormalities.
Navigating as a Deaf and Hard of Hearing Undergraduate in Research Laboratories: Barriers and Inclusion
Authors: Craig Paul A. , Listman Jason , Kurz Kim B. , Picioli Amanda
Published: May 2022

Preparing the next generation of scientists, particularly those from minority groups who are underrepresented in the nation’s scientific workforce, is part of the National Institute of General Medical Sciences’ (NIGMS) mission (NIGMS 5‐Year Strategic Plan Progress and Outcomes, 2020). The Research Initiative for Scientific Enhancement (RISE) Scientists‐in Training Program for Deaf and Hard‐of‐Hearing Undergraduates at the Rochester Institute of Technology (RIT) is an effort to enhance the diversity of the biomedical sciences workforce by providing research‐intensive training for PhD‐bound deaf and hard of hearing (D/HH) undergraduates. RIT‐RISE is designed to support D/HH undergraduate students (known as RIT‐RISE scholars), who navigate academic and science communities differently from hearing students due to various systemic barriers. The D/HH population is unique and diverse in terms of communication and language preferences and identities. Unlike hearing peers, D/HH students often have to request accommodations through the university’s disability office so they can have access to scientific discourse that spontaneously occurs in the research lab. The RIT‐RISE team conducted a qualitative study to better understand D/HH STEM students’ experiences related to communication challenges in undergraduate research environments and to identify best practices to optimize communication access in research environments with mixed D/HH and hearing scientists and STEM students. Based on qualitative data gathered from interviews, focus groups, and lab observations, four main themes emerged from our study: (a) Communication and Environmental Barriers in Research Laboratories; (b) Creating Accessible and Inclusive Lab Environments; (c) Communication Strategies; and (d)Self‐Advocating for Effective Communication. Our findings contribute to the possible strategies to customize and optimize communication experiences and mentoring experiences for all D/HH students.
Molecular investigation of the TTD and PHD histone binding domains of the epigenetic regulator UHRF2
Authors: Albaugh Brittany N. , Ginnard Shane , Winkler Alyssa , Mellado Fritz Carlos , Kemmer Ray , Bluhm Tatum , Butkevich Nick , Gilliam Marisa , Miller Isaak , Bricker Kaitlyn , Basch Madyson , Khuansanguan Panida , Heyl Deborah , Trievel Ray
Published: May 2022

UHRF1 and UHRF2 are epigenetic proteins that regulate both histone and DNA methylation and appear to have associated functions in cancer development. Recently, there has been interest in identifying anti‐cancer molecules that target these proteins. Both proteins contain TTD and PHD histone binding domains that interact with the H3 tail that is trimethylated on lysine 9 (H3K9me3). In this study, we performed a systematic investigation of the isolated TTD and PHD domains of UHRF2, and compared those results with the histone binding properties of UHRF1. We found that while the PHD of UHRF1 is the main binding contributor, the TTD of UHRF2 has a slightly higher binding affinity for H3K9me3. We also determined that a cage of aromatic amino acids in UHRF2 are essential for H3K9me3 engagement and that an asparagine in the H3R2 binding pocket reduces the electrostatic binding potential between the PHD and the H3 tail. Lastly, we show that a unique ~35 amino acid stretch within the TTD of UHRF2 is a disordered region. Together, we offer insights into the mechanistic differences within the TTD and PHD of UHRF1 and UHRF2, which may be potential avenues for identifying drugs that can selectively target these proteins.
Morphological Characteristics of Tubular Polymeric Scaffold to Improve Peripheral Nerve Regeneration
Authors: Dias Fernando J. , Alarcón Apablaza Josefa B. , Lezcano Maria F. , Lopez Alex , Godoy Karina , Oporto Gonzalo H. , Fazan Valéria P. S.
Published: May 2022

Introduction

Peripheral nerve damage causes loss of quality of life due to loss of function. An innovative and promising solution has been the “nerve guide conduits” (NGC) that morphologically must mimic the characteristics of the extracellular matrix to enhance nerve regeneration.

Objective

To gather and describe the main features that should be considered when preparing polymeric NGC to improve peripheral nerve regeneration.

Methods

For this purpose, a Scoping Review was carried out following the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses extension for Scoping Reviews (PRISMA‐ScR) using the following databases: PubMed, Web of Science, Science Direct, and Scientific Electronic Library Online. Only primary studies that analyzed the morphological characteristics of polymeric NGC associated with peripheral nerve regeneration were included.

Results

704 studies were found in the different databases, and only 52 were selected following the inclusion/exclusion criteria. The review of selected studies showed that the polymeric NGC morphological characteristics found were: 1. NGC diameter affects the mechanical properties of the scaffold. 2. Wall thickness of NGC determines the exchange of nutrients, molecules, and neurotrophins between the internal and external environment; and influences the mechanical properties and biodegradation. NGC 3. porosity, 4. pore size, and 5. pore distribution also affects exchanges of substances, their mechanical properties, and biodegradation. The 6. alignment of the NGC fibers influences the phenotype of cells involved in nerve regeneration. And finally, the 7. thickness of the polymeric fiber influences neurite extension and orientation.

Conclusion

Considering the revised literature, a polymeric NGC should consider its diameter adjusted to the nerve with wall thickness, porosity, pore size, and distribution of pores, to favor vascularization, permeability, and exchange of nutrients, and retention of neurotrophic factors, also favoring its mechanical properties and biodegradability.
Obesity Differentially Effects Visceral vs. Subcutaneous Adipose Arteries: Role of Shear Activated Kir2.1 and Alterations to the Glycocalyx in Mediating Endothelial Dysfunction
Authors: Nguyen Thanh , Ahn Sang Joon , Le Master Elizabeth , Lee James C. , Phillips Shane A. , Levitan Irena A. , Fancher Ibra S.
Published: May 2022

Obesity imposes well‐established deficits to endothelial function; however, obesity‐induced endothelial dysfunction is not observed in all vascular beds. Visceral adipose arteries (VAA), but not subcutaneous adipose arteries (SAA), exhibit endothelial dysfunction. Here we aimed to determine if differences in SAA vs. VAA endothelial function observed in obesity are attributed to differential impairment of Kir channels and alterations to the glycocalyx. We used a mouse model of diet‐induced obesity and recruited obese human subjects undergoing planned bariatric surgery to this study. The effects of obesity on flow‐induced vasodilation (FIV) in VAA and SAA were assessed ex vivo. Functional downregulation of endothelial Kir2.1 was accomplished by transducing adipose arteries from mice and obese humans with adenovirus containing a dominant‐negative Kir2.1 construct. Kir function was tested in freshly isolated endothelial cells seeded in a flow chamber for electrophysiological recordings under fluid shear. Atomic force microscopy was used to assess biophysical properties of the glycocalyx. Endothelial dysfunction was observed in VAA of obese mice and humans. Downregulating Kir2.1 blunted FIV in SAA, but had no effect on VAA, from obese mice and humans. Kir shear‐sensitivity was observed in SAA endothelial cells from obese mice and effects on SAA glycocalyx were mild. In contrast, Kir shear‐sensitivity was abolished in VAA endothelial cells and the biophysical properties of the VAA glycocalyx was significantly exacerbated in VAA of obese mice. Our findings show differences in Kir function and alterations to the glycocalyx that we propose contribute to the dichotomy in SAA vs. VAA endothelial function with obesity.
EC Membrane Cholesterol Content Regulates the Contribution of TRPV4 Channels in ACh‐induced vasodilation in Rat Gracilis Arteries
Authors: Salbato Sophia , Morin Emily E. , Walker Benjimen R. , Naik Jay S.
Published: May 2022

Numerous studies show that ion channel activity is sensitive to the level of membrane cholesterol, with cholesterol most often suppressing channel activity. Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48‐hr of exposure to chronic hypoxia (CH). Moreover, CH exposure also couples endothelial Transient Receptor Potential Subfamily V Member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium‐dependent hyperpolarization (EDH)‐pathway not present in control animals. TRPV4 appears to provide activator Ca2+ for endothelial large‐conductance Ca2+‐activation K+ channels in this setting. Taken together, this work suggests that changes in endothelial cell membrane cholesterol mediate this CH‐induced increase in ion channel functionality. Interestingly, both BK and TRPV4 channels contain Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs that bind cholesterol through protein‐sterol interactions. It is essential to separate the effects of CH from the influence of EC membrane cholesterol changes related to hypoxic exposure. Thus, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelial‐dependent vasodilation. To test this hypothesis, vasodilation responses to acetylcholine (ACh) were determined in gracilis arteries from male Sprague‐Dawley rats using standard pressure myography. Rats were exposed to either ambient atmospheric (atm.) pressure or 48‐hrs of hypoxia (0.5 atm). Arteries were pretreated with N‐nitro‐L‐arginine and indomethacin. EC membrane cholesterol was depleted using MβCD and supplemented with MβCD‐cholesterol. In arteries from normoxic animals (a replete cholesterol state), inhibition of TRPV4 did not affect ACh‐induced vasodilation. However, TRPV4 channels contribute to ACh‐induced vasodilation in these arteries when EC membrane cholesterol is depleted with MβCD. In contrast, inhibition of TRPV4 channels attenuated ACh‐induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion with MβCD‐cholesterol in arteries from CH animals abolished the contribution of TRPV4 channels to ACh‐induced vasodilation. In conclusion, our results demonstrate that EC membrane cholesterol impedes the contribution of TRPV4 channels in EDH‐mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.
Anti‐depressant lithium compounds reliefs cardiorespiratory disturbances and improves cardiac function in experimental non‐ischemic heart failure
Authors: Del Rio Rodrigo , Pereyra Katherin , Toledo Camilo , Andrade David C. , Schwarz Karla G. , Diaz Hugo
Published: May 2022

Non‐ischemic heart failure (HF) is a complex medical problem with a high incidence among the older population. While several efforts have been made to improve HF outcomes there is an urgent need for new strategies to reduce mortality rates. Sympathoexcitation and disordered breathing are both considered hallmarks of HF and are closely related to disease progression. Lithium salts has been largely used as anti‐depressants; however, lithium compounds also showed several pleiotropic effects some of them linked to the origin of cardiorespiratory disorders in HF (i.e. oxidative stress). Accordingly, using drug repurposing strategy we aimed to define whether lithium treatment improve cardiorespiratory function in experimental HF. Male Sprague‐Dawley rats underwent volume overload to induce non‐ischemic HF. Four weeks after HF induction, an osmotic minipump containing lithium chloride (Li+, 2 meq/Kg/day) was subcutaneously implanted for drug delivery. Cardiac autonomic and baroreflex function, ventilatory chemoreflex drive and disordered breathing were evaluated after 4 weeks of Li+ treatment in freely moving rats. In addition, cardiac function was determine using PV‐loops. Compared to Sham rats, HF animals displayed autonomic imbalance, reduced baroreflex sensitivity, higher incidence of cardiac arrhythmias, enhanced central chemoreflex and breathing disorders. Cardiac diastolic dysfunction was evident in HF animals compared to Sham animals. HF rats that underwent Li+ treatment showed significant (p<0.05) improvements in cardiac autonomic function as evidenced by reduced low‐to‐high frequency ratio of heart rate variability (LF/HF: 2.8 ± 0.3 vs. 0.9 ± 0.4; HF+Veh vs. HF+Li+), baroreflex sensitivity (BRS: 2.0 ± 0.5 vs. 5.8 ± 1.0 ms/mmHg; HF+Veh vs. HF+Li+) and reduced cardiac arrhythmogenesis (PVC: 220 ± 50 vs. 30 ± 10 events/h; HF+Veh vs. HF+Li+). Furthermore, Li+ treatment in HF completely normalized central chemoreflex drive. Indeed, hypercapnic ventilatory responses obtained in HF+Li+ rats were undistinguishable from the ones obtained in Sham rats (HCVR: 1.7 ± 0.4 vs. 2.0 ± 0.4 DVe/FiCO2; HF+Veh vs. HF+Li+). Also, breathing disorders (i.e. apneas/hypopneas) were markedly reduced by Li+ treatment in HF (AHI: 8.5 ± 1.0 vs. 3.7 ± 0.8; HF+Veh vs. HF+Li+). Lastly, HF+Li+ treated rats displayed marked improvements in cardiac diastolic function (~2.5‐fold reduction in EDPVR) compared to HF+Veh treated rats. We found that the beneficial effects of Li+were partially associated with upregulation of the Nrf2 pathway and related antioxidant genes (i.e. SOD2), reductions in tissue oxidative stress and decreases in the expression of pro‐inflammatory cytokines (i.e. TNF‐a) in HF rats. Our results support the notion that lithium compounds may offer new avenues for the treatment of cardiorespiratory disorders and cardiac diastolic dysfunction in non‐ischemic HF.
Structural and regulatory elements of post‐translational arginylation
Authors: Smith Aaron T. , Van Verna , Ejimogu Nna‐Emeka
Published: May 2022

Eukaryotic post‐translational arginylation, mediated by the family of enzymes known as the arginine transferases (ATE1s), is an important post‐translational modification that can alter protein function and even dictate cellular protein half‐life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cellular division, cytoskeletal function, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. While exploring arginine transferase function, we have discovered that ATE1s bind a previously undiscovered [Fe‐S] cluster. We have used biochemical, spectroscopic, and analytical methods to decipher the composition and reactivity of this [Fe‐S] cluster. Fascinatingly, we find that ATE1 cluster‐binding preserves oligomeric homogeneity while increasing arginylation efficacy, demonstrating that this evolutionarily‐conserved [Fe‐S] cluster regulates arginylation rates. Furthermore, using a combination of X‐ray crystallography and size‐exclusion chromatography‐coupled small angle X‐ray scattering (SEC‐SAXS), we have successfully solved the first atomic‐level structure of an ATE1, which favorably recapitulates its solution‐state behavior. The three‐dimensional structure of ATE1 reveals a bilobed protein containing a GCN5‐related N‐acetyltransferase (GNAT) fold. Structural superpositions, peptide binding, and electrostatic analyses indicate this domain as the location of catalytic arginylation. Additionally, our structure reveals the spatial connectivity of the catalytic machinery to the N‐terminal domain that binds the [Fe‐S] cluster, hinting at the atomic‐level details of the cluster’s regulatory influence. Coupled with a new regulatory framework, the first atomic‐level structure of any ATE1 brings us closer to answering pressing questions regarding the molecular‐level mechanism of eukaryotic post‐translational arginylation.
A Novel Human Heterozygous SCP2 Mutation Leads to Alterations in Lipid Metabolism
Authors: Galano Melanie , Ezzat Shereen , Papadopoulos Vassilios
Published: May 2022

Peroxisomal β‐oxidation plays a critical role in the detoxification of fatty acids and in cholesterol metabolism to form bile acids. The sterol carrier protein‐x (SCPx) is a lipid transfer protein with thiolase activity catalyzing the final step of the peroxisomal β‐oxidation process. SCPx deficiency has been previously reported in only two patients. Here, we report the third patient with SCPx deficiency and the first resulting from a heterozygous mutation in the SCP2gene encoding SCPx. The patient presented with azoospermia, cardiac dysrhythmia, muscle wasting, and progressive brainstem neurodegeneration. The objective of this work was to investigate the link between the patient’s SCP2mutation and his clinical presentations. We hypothesized that, because SCPx plays a critical role in the transport and metabolism of many lipids, the patient’s SCP2 mutation could lead to widespread alterations in lipid‐related genes and their target pathways. Patient fibroblasts (WESP) and normal human dermal fibroblasts (NHDF) were used to investigate the effects of the SCP2 mutation. RNA sequencing was used to identify lipid‐related differentially expressed genes, which led to the identification of metabolic pathways affected by the patient’s SCP2 mutation including cholesterol metabolism, peroxisome proliferator‐activated receptor (PPAR) signaling, and serotonergic synapse signaling. Lipidomic analyses were used to identify changes in the levels of various lipids including several sterols, free fatty acids, phospholipids, and acylcarnitines. This led to the identification of other affected metabolic pathways including steroid biosynthesis, bile acid metabolism, and fatty acid degradation. Collectively, these findings show that the patient’s heterozygous mutation in SCP2 led to alterations in several lipid metabolic pathways, which may explain certain clinical consequences of SCPx deficiency.
Interactions between Fentanyl and 2,5‐dimethoxy‐4‐methylamphetamine (DOM), a 5‐HT2AReceptor Agonist, in Rhesus Monkeys Responding under a Food Versus Drug Choice Procedure
Authors: Maguire David R.
Published: May 2022

5‐HT2Areceptor agonists such as DOM enhance antinociceptive effects of opioids suggesting a 5‐HT2Areceptor agonist could be combined with an opioid to improve treatment of pain. However, it is unclear whether 5‐HT2A receptor agonists also enhance adverse effects of opioids, including positive reinforcing effects that contribute to abuse. This study examined whether DOM alters the reinforcing effects of fentanyl in rhesus monkeys (n=5) responding under a food versus drug choice procedure. Responding on one lever delivered sucrose pellets and responding on the other lever delivered intravenous infusions. Sessions comprised 4 blocks, each with 2 forced trials, during which only one option was available, followed by 6 choice trials, during which both options were available. The unit dose of fentanyl (0.0001‐0.0032 mg/kg/infusion) increased across blocks each session. In the first experiment, sessions were preceded by intravenous administration of DOM (0032‐0.32 mg/kg), naltrexone (0.032 mg/kg), or heroin (0.1 mg/kg). Then, fentanyl was available for self‐administration in combination with DOM in unit dose ratios (fentanyl to DOM) of 1:3, 1:10, or 1:30. Fentanyl increased choice of infusions from less than 10% of trials with the two smallest unit doses to over 80% of trials with the two larger unit doses. Naltrexone decreased choice of fentanyl, shifting the dose‐effect curve rightward, and heroin increased choice of the two smallest unit doses of fentanyl. When administered before the session, DOM reduced choice of fentanyl as well as the number of trials completed. However, DOM did not alter choice or the number of trials completed when available in combination with fentanyl. Attenuation of fentanyl choice by naltrexone and enhancement of fentanyl choice by heroin demonstrate sensitivity of responding to modulation by other drugs. DOM did not appear to enhance the reinforcing effects of fentanyl whether given as a pretreatment or made available in combination with fentanyl. Although pretreatment with DOM decreased choice of fentanyl, it did so at doses that reduced trials completed, suggesting effects of DOM might have been due, at least in part, to generalized rate‐decreasing effects. Taken together with previous studies, these results suggest that DOM enhances some (antinociceptive) but not other (reinforcing) effects of opioids and that combining a 5‐HT2A receptor agonist with an opioid to treat pain would not increase potential for abuse.
Functional analysis of key residues involved in CB1 and CB2 activation
Authors: Brust Christina A. , Ho Jo‐Hao , Stahl Edward , Iliopoulos‐Tsoutsouvas Christos , Nikas Spyros , Makriyannis Alexandros , Bohn Laura
Published: May 2022

Human endocannabinoid signaling is primarily mediated by the G‐protein coupled receptors CB1 and CB2. These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug candidates because of their potential to treat pain and inflammation. Due to their high sequence homology, as well as similarity in activation and signaling mechanism, investigating the physiological role of these receptors with selective ligands has been problematic. Ligands that selectively bind to CB2 may provide therapeutic benefits without the psychoactive effects associated with the CB1 receptor. Recent structural determination of the two receptors has allowed us to identify potential key residues involved in ligand binding. In the studies presented here, single point‐mutations at the CB2 and CB1 receptor were developed and the receptor‐mediated signaling of these mutants were evaluated. Using cannabinoid agonists AM841 and AM12033, we compared each mutation and observed that certain changes did not affect the activity of the receptor while others prevented or severely attenuated signaling. These results may provide a pathway for developing novel CB2 specific compounds with improved therapeutic potential.
Rare Forearm Muscle Variations Seen in Two Cadaveric Dissections
Authors: Sanderson Drew B. , Nation Haley , Rahimi Omid B. , Occhialini Annette
Published: May 2022

Introduction

Anatomical variations of the forearm muscles have been widely reported within the scientific literature, including variations or absence of the extensor pollicis brevis (EPB) and extensor pollicis longus (EPL) muscles. Rare instances have been found of an additional extensor forearm muscle, the extensor carpi radialis accessorius (ECRA). The authors describe a unique extra muscle originating from the muscle belly of the extensor carpi radialis longus (ECRL) and the distal humerus in two cadavers. One of these cadavers also had a complete absence of the EPB and an EPL with two sites of insertion.

Case Presentations

During a cadaveric dissection course, two cadavers were found to have an extra extensor muscle of the left forearm. In both cases this muscle originated from the lateral surface of the ECRL muscle belly and the distal humerus; both muscles had a single tendon that traveled through the first extensor tunnel with the tendons of the abductor pollicis longus (APL) to insert onto the base of the first metacarpal. In addition, in one cadaver the EPB muscle was absent and the tendon of the EPL divided distally to insert into both the proximal and distal phalanx of the thumb.

Discussion

The ECRA is a rare additional extensor forearm muscle described to originate from the humerus below the ECRL and inserts into the first metacarpal bone of the thumb. The ECRA found in each of our two cadavers had an unusual origin from the muscle belly of the ECRL and from the distal humerus superior to the ECRL. This finding adds to the handful of known variations in the ECRA. The EPL and EPB are deep posterior forearm muscles. The EPL usually originates from the middle third of the ulna and inserts onto the base of the distal phalanx of the thumb. The EPB usually originates from the dorsal surface of the interosseous membrane and inserts onto the proximal phalanx of the thumb. In addition to the ECRA found in the left forearm, one of the cadavers also had anatomical variations in the extensor pollicis muscles on the same side. The EPB was absent and the EPL tendon divided distally to insert into the proximal and distal phalanx of the thumb. Variations in the EPB are common, and instances of an absent EPB have been cited in the literature. The EPL variation is most consistent with Turker Classification 1g. It’s possible this represents an anatomical adaptation to compensate for the absent EPB.

Conclusion

While an ECRA muscle is already rare, even more unusual is an ECRA originating from the ECRL and from the distal humerus superior to the ECRL and has not been reported. In addition, the concurrent existence of the three anatomic abnormalities within the same forearm has not been described previously. Knowledge of possible variants in forearm anatomy is crucial for surgeons to have a full understanding of possible causes of pathology. Alternatively, lack of awareness may cause diagnostic confusion or treatment failures.
Timeline of Multi‐organ Plasma Extravasation After Bleomycin‐Induced Acute Lung Injury
Authors: Kitzerow Oliver , Zucker Irving , Lisco Steven , Wang Hanjun
Published: May 2022

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia with the presence of pulmonary edema. ALI is associated with cytokine release and plasma extravasation (PE) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PE. In addition, it is also important to assess PE in the lungs and other organs post‐ALI since ALI/ARDS often causes multi‐organ failure. We hypothesized that ALI induces time‐dependent lung PE, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time‐dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8‐week‐old Sprague Dawley rats to create ALI. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PE. Plasma extravasation of desired organs was evaluated at 3‐, 7‐, 14‐, 21‐, and 28‐days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at the absorption maximum for Evans blue (620 nm). Data show that ALI induces lung PE beginning at day 3 and peaking between 7 and 21 days. Extravasation can also be seen in all organs at varying degrees beginning at day 3 and peaking between day 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time‐dependent PE of the lungs and multiple other organs.
The primary astrocytic mitochondrial transplantation ameliorates ischemic stroke
Authors: Lee Eun‐Hye , Kim Minkyung , Ko Seung Hwan , Lee Minhyung , Park Chang‐Hwan
Published: May 2022

Mitochondria are important organelle which regulate adenosine triphosphate (ATP) production, intracellular calcium buffering, cell survival and apoptosis. They are known to deliver the potential therapeutic role in injured cells through transcellular transfer via extracellular vesicles (EVs), gap junctions, and tunneling nanotubes (TNTs).

Astrocytes secrete numerous factors that promote neuron survival, synapse formation, and plasticity. Recent studies have demonstrated that astrocytes transfer mitochondria into damaged neurons to enhance cell viability and recovery. In this study, we observed that treatment of isolated mitochondria from rat primary astrocytes enhance cell viability and ameliorate H2O2‐damaged neurons. Interestingly, the isolated astrocytic mitochondria increased cell number in damaged neurons but not normal neurons, even though the mitochondrial transfer efficiency was no difference between them. Furthermore, this effect showed in astrocytic mitochondrial transplantation to rat middle cerebral artery occlusion (MCAO) models. These findings suggest that mitochondrial transfer therapy can be used to acute ischemic stroke and other diseases treatment.
Colon Cancer Cell Death by Synergistic Activity of Phytochemicals is Modulated by Increased ROS, Activation of AMPK, and Inhibition of mTORC1
Authors: Srirama Vaishnavi S. , Thomas Rubina H. , Ezekiel Uthayashanker
Published: May 2022

Colorectal cancer (CRC) is the second leading cause of cancer‐related death in the United States, and has high prevalence in both men and women. Consumption of foods high in fruits, vegetables, and spices is strongly associated with a reduced risk of developing cancer, and may be attributable to the synergy of phytochemicals present in the diet. Previously, we have shown that combinatorial treatment with curcumin and silibinin B (CS) led synergistically to higher rates of colon cancer cell death in comparis on to single compound treated cells. Our goal is to elucidate the molecular and signaling mechanisms of the synergistically enhanced anticancer activity mediated by CS in CRC. Cancer cells maintain high energy levels by higher rates of glycolysis and lactic acid fermentation, occurring in the abundance of oxygen, and have elevated levels of reactive oxygen species (ROS) compared to normal cells. Cancer cells adapt to increased levels of ROS by developing ROS scavenging mechanisms. Despite this, the ROS levels are higher than in normal cells. Phytochemicals have been shown to increase ROS by interfering with enzymes involved in reducing ROS. Our hypothesis is that phytochemical‐based disruption of ROS scavenging mechanisms increases ROS to higher levels, which leads to the activation of AMPK. Activated AMPK, in turn, inhibits the mTOR pathway thereby increasing cell death by apoptosis and autophagy. Our initial results showed increased ROS and activated AMPK in combinatorial treated cells compared to single compound and control treated cells.
Brazilian Green Propolis‐Based Nanostructured System: Development, Characterization and its Role in the Modulation of Cytokines in In Vitro and In Vivo Experimental Models
Authors: Beserra Fernando P. , Marincek Andréia , Rodrigues Debora M. , Caldas Gabriel R. , Ferreira Iasmin R. S. , Silva Letícia B. , Schneider Ayda H. , de Sá Keyla S. G. , Gaspary Priscyla D. M. , Bastos Jairo K.
Published: May 2022

Brazilian green propolis from Baccharis dracunculifolia (“alecrim do campo”), stands out for the majority presence of phenolic acids with therapeutic properties well‐described in the literature, such as anti‐inflammatory, antioxidant, and antitumor. However, its direct application, like many natural products, is limited due to its unfavorable physicochemical properties. Thus, we hypothesize that the encapsulation of these compounds in nanostructured systems is a suitable instrument to overcome these limitations. In this work, an oil/water (O/W) nanoemulsion containing Brazilian green propolis extract (NE‐BGP) was developed, and its bioactivity was evaluated in vivo and in vitro experimental models. Quantitative analysis by HPLC showed the presence of coumaric acid, artepelin C and baccharin in the standardized extract obtained, corresponding to 3.2%, 16.8% and 2.3% (w/w), respectively. The developed nanoemulsion showed high stability evaluated for 90 days, with the size around 130 nm, PdI less than 0.3, and negatively charged zeta potential (‐20 mV). The encapsulation efficiency of the markers evaluated in NE‐BGP determined by the validated UPLC‐MS method was superior to 97% for 90 days. Morphological analysis using transmission electron microscopy (TEM) showed globular and spherical shapes. The cytotoxicity was evaluated in 3T3 (murine fibroblasts) and AMJ2‐C11 (murine alveolar macrophages) cells and showed IC50 between 20.88 and 49.32 µg/mL after 24 hours of treatment by the neutral red method, respectively. The in vitro anti‐inflammatory activity in AMJ2‐C11 cells stimulated with LPS 5 µg/mL showed a significant reduction in the levels of nitric oxide (NO), as well as cytokines quantified by CBA (Cytometric Bead Array), IFN‐γ, IL‐4, and IL‐6 in cells treated with NE‐BGP (15 µg/ml) when compared to the control group. Male Balb/C mice (n=5) were used to evaluate the in vivo anti‐inflammatory activity. NE‐BGP administered orally at 100mg/Kg showed a reduction in edema, in the number of inflammatory cells in the joints, and an increase in the mechanical threshold reducing nociceptive stimuli in zymosan‐induced mice arthritis. These results indicate that the developed formulation is an exciting system to encapsulate green propolis extract with high physicochemical stability, anti‐inflammatory and analgesic effects, suggesting NE‐BGP is a possible candidate for the arthritis treatment.
Virtual Circular Genome Model for Prebiotically Plausible Nonenzymatic RNA Replication
Authors: Ding Dian , Zhou Lijun , Szostak Jack W.
Published: May 2022

Nonenzymatic RNA replication is thought to have been an essential step on the path to the emergence of the first ribozymes during the origin of life. However, current nonenzymatic RNA replication models are limited by the extent and fidelity of primer extension chemistry, and the difficulty of copying the strands of the product duplex. Here we discuss the virtual circular genome (VCG) model, which is based on a collection of short oligonucleotides that map onto both strands of one or more virtual circular sequences. Replication is driven by annealing of the oligonucleotides to allow for templated primer extension or ligation, followed by rearrangement to allow additional oligonucleotide elongation. Every oligonucleotide in the system can act as a primer, template, or downstream helper. Therefore, the genomic replication process is distributed across the population. Over multiple generations, this model can be viewed as a flow of material from shorter to longer oligonucleotides that cover the genome. We have monitored the fate of individual primers in the pool of oligonucleotides, and have investigated factors such as oligo length, concentration, temperature, and metal catalyst on the primer extension yield. We propose several strategies that may lead to fast and accurate primer extension in the VCG model, and which may eventually enable cycles of genome replication and the evolution of ribozymes within replicating protocells.
Probing a Proposed Hydroxylation in the Biosynthesis of Bleomycin by Streptomyces verticillus
Authors: Cauthorn Sarah , Calderone Christopher T.
Published: May 2022

Bleomycin is a non‐ribosomal peptide natural product produced by the bacterium Streptomyces verticillus. Non‐ribosomal peptides are produced by non‐ribosomal peptide synthetases (NRPS), which are megaenzymes composed of sequences of modules that incorporate individual amino acids into the non‐ribosomal peptide product. Because amino acids are added to the chain in the order of the modules, we can predict which modules incorporate which amino acids. Bleomycin contains a pyrimidine ring within its structure, the origins of which have not yet been determined. Comparison of the module sequence within the bleomycin NRPS and the structure of bleomycin suggests that asparagine is a precursor to the pyrimidine ring and that it undergoes an oxidation during ring formation. We hypothesize that the oxidation is achieved by hydroxylation of the asparagine to form β‐hydroxyasparagine. This oxidation could occur either on free asparagine or asparagine that had been loaded onto the corresponding NRPS module. We heterologously overexpressed Orf3, a predicted non‐heme iron‐dependent monooxygenases encoded within the bleomycin biosynthetic gene cluster, in E. coli. We examined Orf3's ability to hydroxylate free asparagine and asparagine loaded onto the appropriate NRPS module in vitro via HPLC. We determined that Orf3 did not hydroxylate asparagine in either context. However, we confirmed that asparagine, and not hydroxyasparagine, is loaded onto the NRPS module, confirming that asparagine is a precursor to the pyrimidine ring and strongly suggesting that the as‐yet‐unidentified hydroxylase acts on asparagine after it has been loaded onto the NRPS.