bluuebunny/crossref_metadata_2025 huggingface dataset:
ZNF692, a novel nucleolar regulator in cancer
Authors: Brown Isabella N. , Lafita Navarro M. C. , Hao Yi‐Heng , Conacci‐Sorrell Maralice
Published: May 2022

Nucleolar function and ribosome biogenesis are essential for protein synthesis and cellular growth. Therefore, aberrant increase in nucleolar size and number are hallmarks of many cancers and have been associated with poor prognosis; nucleolar size is one of the most frequently used parameters by pathologists to grade solid tumors. While basic components and general molecular functions of the nucleolus have been studied, fundamental information on the mechanisms and molecules involved in the increased nucleolar size and activity specifically in cancer cells is missing. In an unbiased screen designed to identify growth promoting pathways in MYC‐transformed cells, our lab identified a novel and critical nucleolar regulator, the Zinc finger protein 692 (ZNF692) (manuscript in preparation). Our data has shown that ZNF692 downregulation interferes with protein synthesis and nucleolar function, and overexpression promotes nucleolar activity and protein synthesis. Moreover, we found that ZNF692 expression is dramatically increased in nearly all solid tumors deposited in the TGCA and is correlated with poor patient outcome. On a molecular level, our lab has shown that ZNF692 might play a role in rRNA processing. Through this project, we have unexpectedly discovered that ZNF692 is likely to coordinate cellular and mitochondrial ribosome production to fulfill the needs for rapid cell proliferation. This project aims to define understudied mechanisms and key regulators of increased ribosome biogenesis in cancer cells and could become the basis for the development of novel and unexplored approaches to alter nucleolar activity specifically in solid tumors.
Dissecting the Dual Role of cyclin C in Tumor Suppression
Authors: Willis Stephen D. , Strich Randy
Published: May 2022

Cyclin C has conically been known as a regulator of transcription through its interaction with the CDK8 kinase module (CKM) and the Mediator complex. In response to reactive oxygen species, cyclin C takes on a mitochondrial role, stimulating mitochondrial fission and apoptosis, through its interactions with DRP1 and Bax, respectively. Prior work in our laboratory has identified cyclin C as a tumor suppressor in two murine models of cancer (Thyroid and Pancreatic). In addition, the genomic loci containing cyclin C is lost in many late‐stage cancers. The extent to which the nuclear and mitochondrial roles play in tumor suppression however is unknown. To better understand the significance of these functions on tumor suppression, a system was devised to identify point mutations that separated the transcriptional and mitochondrial function of cyclin C. Using the Sleeping Beauty transposon system, stable cell lines were created by inserting Ccnc variants into Ccnc‐/‐ immortalized mouse embryonic fibroblasts. Cyclin C expression and function were found to be dependent on both promoter choice as well as UTR elements in Ccnc cDNA. Using this expression system, key mutations were found in cyclin C that disabled its transcriptional activity. Going forward these results will be used to address the in‐vivo effects of loss of cyclin C transcriptional activity as it relates to tumor suppression as well as other cellular mechanisms associated with the CKM such as development.
CUIDAME: a pharmacology and nutrition education program for ESPOL workers with Chronic Illnesses
Authors: Orellana‐Manzano* Andrea , Munoz‐Yee Victoria , Vega‐Falconez Noelia , Vizcaino Maria J.
Published: May 2022

Background

Polypharmacy and self‐medication are frequent problems in patients with chronic non‐communicable diseases (CNCD). Accompanied by low health literacy (HA) affects the patient's medical treatment and nutritional status. For this reason, the present study aimed to design a virtual program of good practices for the consumption of medicines and food through digital media to improve the knowledge of these practices in workers of the Escuela Superior Politécnica del Litoral (ESPOL) with NCDs.

Method

The study was descriptive, observational, cross‐sectional, where the Ultra Indispensable Food Care Program for Efficient Medication (CUIDAME) was implemented. Twenty subjects from the areas of administration, teaching, and laboratory technicians from ESPOL participated. These had one or more NCDs and consumed more medications, supplements, or natural products. Updated and personalized information was provided to the educational needs through a Knowledge, Attitudes, and Practices (KAP) questionnaire. The educational material consisted of 4 sets of infographics, two online videos, and one digital guide. With a final survey, knowledge and level of satisfaction were assessed. For the statistical analysis, the InfoStat software was used.

Results

We determined that the participants did not know about their treatment consumption and performed self‐medication. 87% of the participants had not received information on food for medical treatment, 48% had not yet made changes in their diet, but 65% considered that they could improve practices in consuming medications. After the program, it was shown that 53% had adequate knowledge about general dietary recommendations. In general, 59% reflected an excellent, and 41% reflected a very good total rating of the program

Conclusion

Insufficient information about their nutritional therapy and forgetfulness in the medication intake schedule were the leading causes of poor knowledge and lack of treatment adherence. The best strategy for disseminating nutritional and pharmacological information was the CUIDAME Program and the guide given in virtual mode.
Unveiling the Pathologic Response of Cardiac Fibroblasts During Aspergillus fumigatus Pulmonary Infections
Authors: Guirao Abad Jose P. , Jones Shannon , Lowe Elliot , Ivey Malina , Alam Perwez , Askew David S. , Kanisicak Onur
Published: May 2022

Various adverse cardiovascular events have been associated with lung damage as a result of pathological infections followed by chronic inflammation. In this context, several recent studies report severe cardiovascular complications after pulmonary infection by SARS‐CoV‐2. Therefore, determining the physiological link between abnormal pulmonary and cardiac function is an emerging clinical need in the cardiovascular field. The main limitation of these studies has been the lack of specific genetic tools and mouse models to study the cellular and molecular mechanisms that regulate the lung‐heart interactions. To induce lung damage we utilized a common airborne environmental fungus, Aspergillus fumigatus (AF), which is an opportunistic pathogen that establishes pathological infection in the lung if the immune system is compromised. However, in immunocompetent animals, transient stress to the lung is still detectable before clearance via immune cells. Our lab has discovered that pulmonary fibroblasts (PFs), typically associated with the maintenance and production of the extracellular matrix, activate in response to inhaled AF spores and modulate the immune response. Moreover, we have also observed activation of cardiac fibroblasts (CFs) in response to the pulmonary AF infection. Therefore, we hypothesized that pulmonary infection leads to cardiac stress, resulting in the activation and pathological response of CFs, and subsequently result in myocardial remodeling, fibrosis, and disease. To test our hypothesis we used our recently developed and described in vivo cre‐loxP lineage tracing mouse lines to specifically tag and manipulate all activated fibroblast during infection, clearing and reinfection. Our data show that CFs can activate after an acute inhalation of AF spores which are quickly cleared in immunocompetent mice with no pathological lung damage. Moreover, subsequent asymptomatic chronic AF infections result in the accumulation of PF and CF pathologic activation. These results reveal a cellular novel link between pulmonary infection and its impact on cardiovascular events.
New genetically‐encodable tools to probe the nuanced regulation of the Extracellular signal‐regulated kinase (ERK) pathway
Authors: Keyes Jeremiah , Romania Taylor , Boaks Adam , Kotau Aliaksandr , Baldwin Peter , Slye Kevin
Published: May 2022

The Extracellular signal‐regulated kinase (ERK) signaling axis is activated by a host of divergent signaling molecules which each induce their own specific responses. These cellular responses include fundamental processes such as proliferation, differentiation, migration, and, under certain conditions, even apoptosis. Thus, the ubiquitous utilization of ERK signaling in variable and contrasting cellular processes makes it difficult to inhibit the pathway without causing adverse side effects. Furthermore, dysregulation of the Ras/Raf/MEK/ERK signaling pathway is directly implicated in a variety of disorders ranging from cancer to diabetes. Here, we report new tools to elucidate the nuanced mechanisms of differential regulation of the ERK pathway. First, we report a new genetically‐encodable tool to inhibit ERK at specific subcellular locations, allowing us to explore the specific function and mechanisms of regulation of ERK with extremely precise subcellular resolution. Additionally, we report a new genetically‐encodable fluorescent biosensor based on previous generations of the ERK‐kinase activity reporter (EKAR). Our new biosensor, which we have termed reDox‐EKAR1, allows us to probe into the spatiotemporal activity of ERK towards a different class of substrates than substrates probed by previous generations of EKAR. Utilizing these new tools, we report how specific pools of ERK regulate particular cellular functions previously underappreciated in the field, and we present our preliminary results detailing how these specific subcellular pools of ERK are differentially regulated.
Project 80: a reproducible, student‐driven framework for creating multimedia educational resources from primary literature
Authors: Liu Houjun , Krishnan Anoushka , Brown Micah , Hauser Paul , De Luke
Published: May 2022

Natural science education resources traditionally teach only codified theory. While theory education is crucial, much of academic science takes place via scrutinizing contested scientific discourse. Due to such resources’ content complexity, high school students are rarely exposed to current, debatable, and relevant science. In response, we introduce Project80: a systemic, student‐run protocol to synthesize the latest primary literature in a sub‐field into approachable, produced multimedia educational content. The protocol is run by a team of 7 students over the course of 1 month. Students running the protocol consume complex scientific literature, distill relevant data and findings, and synthesize a culminating product of audiovisual content to supplement existing biology and chemistry pedagogy. The system runs independently with limited faculty involvement. Our analysis indicates that the multimedia content created by this protocol will be relevant to roughly 30 courses locally at our institution and will have further extensions in secondary education beyond.
Endothelin‐1 receptor antagonists reduce HbA1c in obese patients with pulmonary hypertension
Authors: Stapel Jennifer , Speed Joshua S. , Clemmer John S.
Published: May 2022

Obesity is associated with insulin resistance and type II diabetes. Our lab has recently shown that blockade of endothelin‐1 (ET‐1) receptors attenuates insulin resistance in a mouse model of diet induced obesity. Currently, ET‐1 receptor blockers are FDA approved to treat patients with pulmonary hypertension. We hypothesized that patients taking ET‐1 receptor antagonists (ERA) have improvements in insulin sensitivity. Electronic health record data were extracted from EPIC (Epic) at the University of Mississippi Medical Center. Data from 2013‐2020 were retrieved from patients ≥18 years old with a clinical diagnosis of pulmonary hypertension and at least two clinical visits within 2 years. Patients that were prescribed ERAs (n=12) were similar in age (61±14 vs. 60±14 yr), BMI (34±8 vs. 35±11 kg/m2), diabetes prevalence (73% vs. 80%, p=0.59), and follow‐up time (209±74 vs. 283±180 days) as compared to control patients (n=137). Additionally, there was a similar decrease in BMI at follow‐up in the control (‐1.6±5 kg/m2) and ERA patients (‐1.9±3 kg/m2). Interestingly, at follow‐up, HbA1c significantly decreased ‐12±11% in patients taking ERA while control patients were associated with a 2±20% increase in HbA1c. In the ERA patients, circulating LDL and total cholesterol tended to decrease (‐6±23% and ‐6±13%, respectively) and HDL tended to increase (‐9±32%) after ERA but did not reach statistical significance. These data suggest a potential anti‐diabetic effect of ERAs in patients with obesity and insulin resistance.
Effects of Creatine Nitrate and Caffeine Alone and Co‐ingested on Anaerobic and Muscular Endurance Performance
Authors: Koozehchian Majid S. , Mabrey Gina , Newton Andrew T. , Forbes Scott C. , Naderi Alireza , Haddad Monoem
Published: May 2022

BACKGROUND

Ingestion of creatine nitrate and caffeine individually improve anaerobic and muscular endurance performance; however, co‐ingestion of creatine nitrate and caffeine remains unclear.

METHODS

In a double‐blind, randomized, placebo‐controlled cross over study, 12 resistance‐trained participants (22±1 y; 180.0±9.5 cm; 85.9±15.1 kg) completed 4 conditions in random order: creatine nitrate (CN: 5 g/day + 0.675 g/day of maltodextrin), caffeine (CAF: 400 mg/day + 5 g/day of maltodextrin), CN+CAF (5 g/day of creatine nitrate + 400 mg/day of caffeine), and placebo (PL: 5.4 g/day of maltodextrin). Supplements were ingested for 7 days with a 7‐day washout period between conditions. Before and after supplementation, muscular endurance (bench press and leg press; 3 sets at 70% 1RM) and a Wingate anaerobic test were performed. Peak power (PP), mean power (MP), and fatigue index (FI) were calculated. Data were analyzed using a two‐way (within‐within) analysis of variance (p≤0.05) with a Tukey’s post hoc when appropriate. Absolute change (post minus pre‐testing) was calculated and analyzed with a repeated‐measures ANOVA.

RESULTS

There was a significant main effect of time for both leg press (p=0.009) and bench press (p<0.001) with post‐testing greater than pre‐testing; however, there were no differences between conditions. For peak power, there was no significant interaction (p=0.436), the main effect of time (p=0.967), or differences between conditions for absolute change (p=0.436). For mean power, there was a significant interaction (p=0.037); however, post hoc analysis revealed no significant pairwise effects; however, absolute change for mean power between conditions was significant (p=0.037), with post hoc analysis revealing a trend (p<0.10) for both CN conditions (CN alone and CN+CAF) to be superior compared to CAF. There were no significant differences between conditions or overtime for fatigue index.

CONCLUSION

Short‐term CN and CAF ingested either individually, or co‐ingested could not augment upper or lower body muscular endurance or anaerobic performance compared to PL in resistance‐trained participants.
Expression profile of miR‐148a and miR‐155 associated with apoptosis mechanism in pancreatic cancer
Authors: Novais Paulo C. , Moscatel Matheus B. , Rosa Marcella S. , Tirapelli Daniela , Cirino Mucio L. , Kemp Rafael , Santos José S.
Published: May 2022

Pancreatic adenocarcinoma (PA) is present in 90% of pancreatic neoplasms. Risk factors such as age, gender, and ethnicity, among others, should be considered for the involvement of cancerous lesions. Most patients with PAat the time of diagnosis, have locally advanced disease or distant metastases. The resectability of the PA is generally less than 20% and the annual incidence rates are equivalent to those of mortality, which means a survival rate of less than 5% in five years. Studies show that PA presents a series of molecular alterations associated with its tumor malignancy, among the most studied, apoptosis stands out. Many studies also demonstrate the important role of microRNAs in the regulation of anti and pro‐apoptotic targets. MicroRNAs are non‐coding RNAs (ncRNAs) consisting of 19 to 25 nucleotides, they are small endogenous molecules and act by controlling gene expression in several pathological processes, such as neurological degeneration, autoimmune diseases, inflammatory processes, diabetes, and cancer. The aim of the present study was to analyze the expression of microRNAs miR‐148a and miR‐155 in pancreatic adenocarcinoma. MiR‐148a and miR‐155 expression were analyzed in 10 samples from patients diagnosed with pancreatic adenocarcinoma from surgical resections and 5 control samples from healthy donors using the RT‐PCR technique. Based on the results obtained in our study, we can conclude that although no statistically significant difference was observed between the groups, a lower expression of miR‐148a and miR‐155 (pro‐apoptotic) can be observed in the PA group when compared to control group.
The role of physical exercise in apoptosis in an experimental cerebral ischemia model
Authors: de Souza Gabriel P. N. , Porsani Lucas B. , Tirapelli Victor C. , Stevanato Ana Laura R. , Novais Paulo C. , de Assis Cirino Mucio L. , Tirapelli Daniela , Tirapelli Luis F.
Published: May 2022

Introduction

Stroke is a major public health problem, with high mortality rates and high levels of disability. Between 1990 and 2010 for example, stroke increased from the fifth to the third leading cause of disability. In addition, its incidence has increased among younger people, a situation in which there are significant health consequences and elevated social costs. There is evidence that physical exercise promotes neuroprotective effects when used as a therapeutic treatment in an experimental ischemia model. However, the mechanisms of neuroprotection are still not well understood.

Objective

The objective of this study was to evaluate the expression profile of the microRNAs miR‐21, miR‐16, miR‐155 and caspase‐3 and Bcl‐2 genes by real‐time PCR in brain tissue (ischemic focus) of the animals after the performance of physical training before focal cerebral ischemia.

Materials and Methods

48 Wistar rats were used. The animals were divided into four experimental groups: control group, ischemia group, exercise group and exercise + ischemia group. The real‐time PCR methodology was used to analyze the expression of miRNAs; miR16, miR‐21, miR‐155, caspase‐3 and Bcl‐2.

Results and conclusions

In our study, we did not observe statistically significant differences in the expression of miRNAs: miR‐16, miR‐21, miR‐155 or in the Bcl‐2 and caspase‐3 genes in brain tissue in the groups undergoing cerebral ischemia and exercise when compared to the control group.
Measuring AMPK Activity in Vivo Using a Genetically‐Encoded Biosensor
Authors: Carman Lynly , Nelson Matthew D.
Published: May 2022

Adenosine monophosphate‐activated kinase (AMPK) is a highly conserved heterotrimeric protein complex that functions in a broad spectrum of cellular stress response pathways. Most work investigating the cellular dynamics of AMPK activity has been limited to either whole‐organ/tissue or, in the case of smaller genetic models like Caenorhabditis elegans, whole‐organism analyses. Some limitations of these approaches include: 1) An inability to observe cellular heterogeneity of AMPK activation; 2) a lack of the temporal dynamics; 3) an inability to correlate AMPK dynamics with physiological states of a living organism. Thus, we sought to adapt a genetically‐coded AMPK biosensor, called AMPKAR‐EV (Kongaya et al., 2017), for use in C. elegans, which is transparent and genetically tractable. The AMPKAR‐EV biosensor uses fluorescence resonance energy transfer (FRET) to report AMPK phosphorylation of substrates. We expressed codon‐adapted AMPKAR‐EV in C. elegans and made three separate transgenic lines expressing it in intestines, body wall muscles, and neurons. As expected, we find that the biosensor responds to conditions that promote AMPK activation. We crossed the lines expressing AMPKAR‐EV in the intestines into a number of different genetic mutants, which we either generated using CRISPR or obtained from previous studies. For context, work in mammals suggests that protein kinase A (PKA) phosphorylates AMPK at serine 173 (S173 ‐ inhibitory site) and prevents catalytic phosphorylation at threonine 172 (T172 ‐ activation site). These sites are conserved to C. elegans but located at serine 244 (S244) and threonine 243 (T243) respectively; however, the significance of these regulatory sites have not been specifically studied. To test these mechanisms more precisely, we made two C. elegans strains that also express AMPKAR‐EV, one that alters the activation site (aak‐2(T243A)) and another that alters the inhibitory site (aak‐2(S244G)). The third strain we constructed expresses AMPKAR‐EV in the kin‐2(ce179) mutant background, which exhibits constitutively active PKA. Using these strains we are optimizing AMPKAR‐EV in vivo and testing the hypothesis that PKA inhibits AMPK at the S244 inhibitory site, which prevents its phosphorylation at the activation site. We envision that AMPKAR‐EV will be of broad use in C. elegans as an organism well‐known for its study of behavior, aging, and longevity.
Scaling of Antibacterial Immune Defenses in Mammals
Authors: Downs Cynthia J. , Schoenle Laura A. , Goolsby Eric W. , Oakey Samantha J. , Ball Ray , Jiang Rays H. , Martin Lynn B.
Published: May 2022

Terrestrial mammals span 7 orders of magnitude in body size, ranging from the < 2 g pygmy shrew (Suncus etruscus) to the > 3900 kg African elephant (Loxodonta africana). Although body size profoundly affects the behavior, physiology, ecology, and evolution of species, how investment in immune defenses changes with body size across species is unknown. Comparative immunology studies are often limited because of reagents or because assays are calibrated for a specific species. We developed a novel 12‐point dilution‐curve approach to compare complement‐mediated antibacterial capacity against 3 diverse bacterial species (Escherichia coli, Salmonella enterica, Micrococcus luteus) among >160 terrestrial species of mammals. We tested published predictions about the scaling of immune defenses, focusing on the Safety Factor Hypothesis, which predicts that broad, early‐acting immune defense should scale hypermetrically with body mass. Multivariate linear mixed models using Markov chain Monte Carlo techniques demonstrate that antibacterial activity across mammals exhibited isometry (scaling coefficient = 0), regardless of the bacterial species used in the challenge. For example, the antibacterial ability of a specific dilution scaled isometrically when serum was tested against E. coli(b95% Credible Intervals =‐0.144 : 0.044), S. enterica (b = ‐0.102 : 0.061), and M, luteus (b = ‐0.401 : 0.177). Therefore, body size was unrelated to killing capacity across species. Intriguingly, these results indicate that the serum of large mammals was less hospitable to bacteria than would be predicted by their metabolic rates. That is, if metabolic rates restrict the rate of organismal reactions as postulated by the Rate of Metabolism Hypothesis, large species should have disproportionately lower antibacterial capacity than small species, but they do not. These results have direct implications for effectively modeling the evolution of immune defenses and identifying potential reservoir hosts of zoonotic pathogens. This work was supported by the National Science Foundation (award numbers IOS 1656551 to C.J.D. and IOS 1656618 to L.B.M), and the authors have no competing interests to declare.
Impaired Endothelial Function and Enhanced Contractility of Microarterioles from Persons Living with Human Immunodeficiency Virus (HIV+) is Mediated by Inflamed Perivascular Adipose Tissue
Authors: Wilcox Christopher S. , Wang Dan
Published: May 2022

This research investigates the mechanisms that contribute to the enhanced cardiovascular disease (CVD) in HIV+ persons. It tests the hypothesis that perivascular adipose tissue (PVAT) mediates the microvascular dysfunction. Subcutaneous microarterioles were dissected from gluteal skin biopsies from 11 HIV+ and 10 matched control women aged < 50 years who were free of CVD risk factors (except increased BMI). Arterioles were mounted on isometric myographs with or without PVAT. Nitric oxide (NO) and reactive oxygen species (ROS) were measured by fluorescence microscopy. PVAT significantly (P < 0.05) enhanced endothelium‐ dependent relaxation (EDR) and NO with acetylcholine and moderated contractions with thromboxane and endothelin 1 (ET1). But these effects of PVAT were significantly (P < 0.05) diminished in HIV+ vs control subjects. Although microarterioles from HIV+ had enhanced ROS with ET1. ROS was not affected by PVAT. Incubated PVAT from HIV+ persons released significantly (P < 0.05) more leptin (47 +/‐ 8 vs 22 +/‐ 3 ng · mg‐1) and less adiponectin (3.0 +/‐ 0.4 vs 4.6 +/‐ 0.5 µg · mg‐1) but more 8‐isoprostane F2α (2.5 +/‐ 0.4 vs 1.3 +/‐ 0.3 µmol · mg‐1) and inflammatory cytokines such as TNFα (11 +/‐ 1 vs 5 +/‐ 1 pg · mg‐1).

Conclusion

PVAT from young HIV+ women has enhanced oxidative stress and inflammation and impaired ability to enhance microarteriolar NO and moderated contractility. Thus, PVAT and adipokines should be novel foci for therapy to reduce CVD in HIV+ persons.
Myristoylated Protein Kinase C Beta II Inhibitor Attenuates Renal Injury in Mice Subjected to Severe Bilateral Ischemia‐Reperfusion
Authors: Shah Lisa , Johnson Devani , Verwoert Alexis B. , Singh Sunit G. , Dean Tameka C. , Chen Qian , Barsotti Robert , Jiang Yanlin , George James , Agarwal Anupam , Young Lindon
Published: May 2022

Acute kidney injury (AKI) due to ischemia‐reperfusion (I/R) insult involves oxidative stress and inflammation leading to rapid renal decline and remains a significant cause of post‐operative mortality. Myristoylated protein kinase C beta II peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) is known to attenuate myocardial I/R injury in ex vivo rat hearts. We hypothesized that myr‐PKCβII‐ would attenuate severe renal I/R injury that is characterized by elevated serum creatinine (Cr) and a decrease in glomerular filtration rate (GFR). We predict that treatment with myr‐PKCβII‐ will improve these indices of kidney function compared to a scrambled control peptide (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram).

Renal pedicles of anesthetized male C57BL/6J mice (25–30g) were clamped bilaterally for 20 min or 19 min. Five minutes before unclamping, 2.0 mg/kg (20 µM serum) myr‐PKCβII‐ or myr‐PKCβII‐scram were given i.v. into the tail vein. Cr (mg/dL) was measured at baseline, 24h, 72h, and 96h post‐injury. GFR (µl/min) was determined with fluorescein‐isothiocyanate (FITC)‐Sinistrin renal clearance. Data were evaluated by unpaired Student’s t‐test.

Following 20‐min renal ischemia (Fig 1.), myr‐PKCβII‐ (n=9) significantly reduced Cr at 24h and 72h post‐injury compared to myr‐PKCβII‐scram control (n=8, p<0.05). However, there were three fatalities prior to 96h. In 19‐min ischemia, there were no fatalities up to 96h post‐injury. However, Cr levels of the myr‐PKCβII‐scram control (n=8) in 19‐min I/R were significantly lower than 20‐min I/R at all time points post‐injury (all p<0.01): 24h (0.54 ± 0.10 vs 1.59 ± 0.06 mg/dL), 72h (0.24 ± 0.06 vs 1.28 ± 0.25 mg/dL), and 96h (0.17 ± 0.03 vs 0.72 ± 0.15 mg/dL). Myr‐PKCβII‐ did not significantly reduce Cr (Fig. 1) nor improve GFR (Fig. 2) following 19‐min I/R.

Results suggest that 20‐min renal ischemia was more severe, which was indicated by a 5‐fold increase of peak Cr levels at 72h post‐injury compared to 19‐min ischemia and the unanticipated fatalities of three mice. Myr‐PKCβII‐ attenuated renal injury following 20‐min renal ischemia, but not at the milder 19‐min model in which peak Cr levels were too low to detect therapeutic benefit. The large difference in injury severity between 20‐min and 19‐min renal ischemia emphasizes the temporal relationship between renal function and ischemic duration.

Future studies will characterize myr‐PKCβII‐ protection against AKI in 19‐min bilateral renal ischemia based on biomarkers indicating proximal renal tubule damage (e.g. NGAL and Kim‐1). A more optimal ischemic duration between 19 min and 20 min will be investigated for future experiments.
Oxytocin, Prostaglandin F2α, and Prostaglandin E2 Induced Contractions in Isolated Uterine Smooth Muscle Strips from Non‐pregnant, Late Pregnant, and Postpartum Rats
Authors: Beard Jenifer M. , Miknich Kaitlin B. , Sarkis Mina Nader , Gusek Brikena , Jones Carleton (. , Eckman Delrae M.
Published: May 2022

Objective/Introduction

Numerous studies have investigated uterotonic agents (e.g., oxytocin) in uterine smooth muscle (USM) from non‐pregnant (NP) and late‐pregnant (LP) women and/or animal models of pregnancy. Surprisingly, few studies have investigated USM from the post‐partum period to determine whether USM contractility returns to its pre‐pregnant state. We designed a study in rodents to investigate whether USM contractility to oxytocin (OXY), prostaglandin F2α (PGF2α), and prostaglandin E2 (PGE2) returned to pre‐pregnant values within 30 days postpartum.

Methods

Studies were performed in NP (n=12), LP (21d gestation, n=4), 3 days post‐partum (PP3, n=2), and 30 days post‐partum (PP30, n=4) Sprague Dawley rats. Uterine horns were cleaned of connective tissue and cut along the longitudinal axis allowing isolation of USM (width: 2mm, length: 4±1mm) oriented in the longitudinal muscle (LM) or circular muscle (CM) direction. CM or LM strips were mounted in tissue baths containing Krebs and equilibrated @ 37°C for 60 min prior to starting one of the two following protocols.

Protocol 1 identified the optimal resting tension (in grams: 0.25, 0.50, 1.0, and 1.5) to measure spontaneous contraction (SCA), K+‐induced contraction (60mM K+), and cumulative OXY‐induced contraction (10‐11 to 10‐7) in CM and LM of NP rats (N=4‐6).

Protocol 2 was designed assess SCA, K+‐induced contraction, cumulative concentration‐response relationships to OXY (10‐11 to 10‐7), PGF2α (10‐8 to 10‐4), or PGE2 (10‐8 to 10‐4) in CM and LM, of NP, LP, PP3, and PP30 rats (N=2‐6).

Statistical comparisons were made using either a t‐test or two‐way ANOVA, and considered statistically different at P<0.05.

Results

Protocol 1‐ LM SCA was greater than CM SCA at all resting tensions. K+‐induced constrictions in LM and CM were similar at each resting tension. OXY‐induced contraction in LM was generally greater than CM (P<0.05 at 0.25g, 0.5g, and 1.5g tension, comparable at 0.5, 1.0, and 1.5g tension).

Protocol 2 ‐ SCA in NP, LM was greater than NP, CM (P<0.05). There was no difference in SCA in CM vs LM in USM from LP, PP3, and PP30 animals. K+‐induced constrictions in CM and LM were similar in USM from NP, LP, PP3 and PP30 animals. OXY‐induced contractions were greater in LM than CM for all pregnancy states (P<0.05). PGE2 contractions were greater in LM vs CM in USM from NP rats (P<0.05), but were similar in USM from LP, PP3, and PP30 animals. In contrast, PGF2α‐contractions were similar in LM and CM for all pregnancy states.

Summary/Conclusion

Our preliminary data suggests that uterotonic agents be assessed at either 0.5g or 1.0g resting tension. In addition, uterotonic agents exhibit significantly greater contraction in longitudinal vs circular orientation. Finally, contractility to uterotonic agents returns to pre‐pregnant values by PP30.
The Effect of Leucine and Doxorubicin Treatment on Skeletal Muscle Mitochondrial Function
Authors: Carlen Austin E. , Marchant Erik D. , Evans Richard W. , Hancock Chad R.
Published: May 2022

Doxorubicin (DOX) is a common, effective chemotherapy drug. However, DOX treatment causes the negative side‐effects of skeletal muscle atrophy and weakness. One of the key mechanisms by which DOX can lead to increased atrophy is through increased mitochondrial damage and inhibition of protein synthesis via down‐regulation of mTOR. Leucine (LEU), an essential branched‐chain amino acid, activates mTOR, increases mitochondrial biogenesis, and muscle protein synthesis. It is not clear if enhanced activation of mTOR by LEU might moderate the damaging effects of DOX in skeletal muscle. The purpose of this study is to determine if LEU supplementation attenuates the loss of mitochondrial function caused by DOX. C2C12 myoblast cells were grown to confluence and then treated with 10 mM LEU, 0.5 μM DOX, or both for 24 hours. High‐resolution respirometry was performed on cells using the Oroboros O2K respirometer to assess mitochondrial function in leak, coupled, and uncoupled states. Total cells used for respiration were assessed by aliquoting a portion of cells before respirometry and performing a BCA protein assay on this representative aliquot. Citrate synthase activity was also evaluated on cells used for respirometry as a marker of mitochondrial content. We report that DOX caused reduced leak, maximum coupled respiration, and uncoupled respiration by 33%, 42%, and 38%, respectively (p<0.05). DOX also lowered citrate synthase activity by 44% (p<0.05). Total protein, measured for total cell homogenates was reduced by 63% with DOX treatment (p<0.05). Contrary to our hypothesis that LEU may mitigate the increase in cellular degradation seen with DOX treatment, there was no significant difference between DOX treatment and DOX + LEU treatment. These findings suggest DOX interferes with LEU’s ability to upregulate mTOR activity. Interestingly, the total protein content was reduced significantly more by DOX (63%) than either reductions seen in mitochondrial respiration (~40%) and citrate synthase activity (44%). These findings suggest that the effects of DOX on general cellular protein content is far greater than the negative effects observed on mitochondrial content or function.
Complex II‐linked mitochondrial respiration is upregulated during postnatal development in high‐altitude grown mice
Authors: Arias‐Reyes Christian , Aliaga‐Raduán Fernanda , Rollano‐Peñaloza Oscar M. , Iturri Pablo , Soliz Jorge , Joseph Vincent
Published: May 2022

Recently, we have shown that chronic normobaric hypoxia (12% O2 – 21 days) triggers mitochondrial and cell‐ metabolic plasticity in the retrosplenial cortex of male adult FVB mice but not in SD rats. In these animals we reported a transient upregulation of anaerobic metabolic pathways (glycolysis + lactate metabolism) followed by an attenuation in the mitochondrial respiration. These results pointed to an optimized use of oxygen at cellular level in an area of the brain responsible of processing visual information and spatial memory. Features of great importance in the biological fitness. In the current work we investigate how the residence in high altitude (La Paz – Bolivia, 3,600 m) affects the developmental profile of the mitochondrial respiratory efficiency in the retrosplenial cortex of mice and rats. We hypothesize that the mitochondrial plasticity observed in adult mice will have a process of maturation during postnatal development. This response shall be absent in rats.

To do so, we measured the mitochondrial oxygen consumption rate (OCR) in saponin‐permeabilized brain samples from FVB mice and SD rats born and grown in La Paz. The animals were sacrificed at postnatal ages p7, p14, p21, and adulthood (P90), and the OCR linked to the use of NADH (N pathway), FADH2 (S pathway), or both (NS pathway) was quantified by means of high‐resolution respirometry (OROBOROS).

Our results show a pattern of maturation (increase) of the mitochondrial respiration in the retrosplenial cortex along with the postnatal development (21 days+) of mice and rats. However, an upregulation of the participation of the S pathway (complex II‐linked) in the mitochondrial respiration was observed during the first three weeks of age only in mice. This observation supports previous works suggesting a key role of the complex II and succinate (substrate of complex II) in the successful cellular and mitochondrial acclimatization to hypoxia. These findings contribute to further establish FVB mice and SD rats as a model to study divergent acclimatization to hypoxia.
Comparative Studies on the Interaction of Heparins from Various Origins with Heparin Cofactor II and Antithrombin
Authors: Baig Nausheen , Kouta Ahmed , Jaradeh Mark , Hoppensteadt Debra , Jeske Walter , Fareed Jawed
Published: May 2022

Introduction

Unfractionated heparins (UFH) of mucosal origin are currently obtained from bovine, ovine, and porcine sources. In the USP potency assays, bovine mucosal heparin (BMH) exhibit much lower activities (140 U/mg) in comparison to porcine mucosal heparin (PMH; 195 U/mg) and ovine mucosal heparin (OMH; 200 U/mg). Besides molecular and structural differences, these heparins have differential interactions with serpins such as antithrombin (AT) and heparin cofactor II (HCII). The purpose of this study is to compare the inhibitory profiles of various heparins in systems enriched with AT and HCII.

Materials and

Methods

The active pharmaceutical ingredients (API) of BMH, OMH, and PMH were obtained from Kin Master (Brazil), Ronssi (China), and Medefil (USA). Bovine (B.LMWH), ovine (O.LMWH), and porcine (P.LMWH) derived low‐molecular‐weight heparins (LMWH, enoxaparin) were obtained from Ronssi (China). Purified human AT, HCII, and thrombin (IIa) were obtained from Enzyme Research (USA). All heparins (UFH’s and LMWH’s) were supplemented to AT and HCII enriched buffer systems at various gravimetric and potency equated concentrations. The relative inhibition of IIa was measured in terms of percent inhibition using an amidolytic method employing IIa specific substrates. Percent inhibition was calculated for each concentration in both systems for each of the three heparins. Inhibitory concentrations required to inhibit 50% of thrombin (IC‐50) was calculated for each UFH and LMWH in terms of µg/mL and U/mL.

Results

In the AT supplemented system using gravimetric concentrations of various UFH’s, PMH and OMH showed comparable values, whereas BMH exhibited much higher IC‐50’s. However, the LMWH’s of various origins showed comparable IC‐50 values. In this same system, the USP potency adjusted heparins exhibited comparable results for all UFH’s (Figure 1). In the HCII supplemented system, UFH’s of various origins exhibited similar IC‐50 values in both gravimetric and USP potency adjusted studies. Similarly, the LMWH’s when compared in gravimetric amounts demonstrated similar IC‐50 values (Figure 2). The relative IC‐50 values of the LMWH’s in both the AT and HCII enriched systems were higher than the UFH’s.

Conclusion

These results show that potency adjusted UFH’s exhibit comparable inhibition of IIa in AT and HCII enriched systems. The differing BMH results observed in these two systems at gravimetric concentrations may partly be due to differences in charge density and AT‐binding consensus sequences. The depolymerized enoxaparin of different origins did not reveal any significant differences in the IC‐50 values. This may be due to the chemical modifications that occur during the manufacturing of these agents. Future studies may be warranted to further investigate these cofactors and their impacts on the function of various heparins.
Role of HCN Currents in Epileptiform Activity in Subicular Neurons
Authors: Alfred Monica , Sikdar Sujit K.
Published: May 2022

Subicular neurons play a critical role in Temporal Lobe Epilepsy. While studies have shed light on how synaptic conductances contribute to epileptiform activity in these neurons, it is still unclear how intrinsic conductances influence epileptic discharges. HCN channels play crucial role in pathophysiology by modulating the electrophysiological properties of neurons. To investigate the ability of intrinsic properties in a subicular neuron to generate epileptiform activity, we studied the effects of Ih (HCN) currents in 4‐Aminopyridine magnesium‐free epileptic model in various classes of subicular neurons. To study the modulation of epileptiform activity due to Ih in these neurons, we modeled subicular HCN currents in dynamic clamp that mimicked the effects of blocker and overexpression. We observed similar changes in input resistances with the subtraction and addition of currents in burst firing, regular firing and interneurons. However, the firing rate characteristics during epileptiform activity with the modulation of HCN channels in burst firing and regular firing differed from those observed in interneurons. The results indicated that the burster cells majorly contribute to the epileptic firing characteristics due to HCN in subiculum.

During the epileptiform activity, these bursters showed progressive loss of homeostatic compensation characterized by decrease in sag ratio and and loss in theta frequency. The loss of homeostatic compensation incurred was found to be irreversible. These irreversible changes are also characterized by altered HCN channel kinetics. To study how these epilepsy‐induced alterations govern neuronal excitability, we studied the electrophysiological profiles of subicular neurons with altered HCN current kinetics introduced through dynamic clamp in the presence of ZD7288. These irreversible changes are also characterized by altered HCN channel kinetics. To study how these epilepsy‐induced alterations govern neuronal excitability, we studied the electrophysiological profiles of subicular neurons with altered HCN current kinetics introduced through dynamic clamp in the presence of ZD7288. The results showed that the theta selectivity and sag profiles were modulated and post‐inhibitory rebound firing were evoked making the cell hyperexcitable. It is known that the stable neuronal network is homeostatically regulated by intrinsic excitability and synaptic currents. Studies are being conducted to understand the interaction between Ih and synaptic currents before and during the epileptiform activity in neurons with post‐epileptic induced hyperexcitability.
A Novel Animal Model in Diabetes: Regeneration of β cells in the Axolotl Salamander?
Authors: Sørensen Pernille L. , Dittrich Anita L. , Lauridsen Henrik L.
Published: May 2022

Diabetes is an ever‐growing health issue affecting an estimate of 463 million people worldwide. Type 1 diabetes is characterized by hyperglycemia and loss of pancreatic β cell mass. Currently, there is no curative treatment of β cell loss. Humans have a poor natural ability to regenerate β cells, empathizing the importance of conducting research in regenerative medicine. However, this field of study is limited by the animal models available due to their inadequate intrinsic ability to regenerate, size and/or remoteness from humans. As the axolotl salamander (Ambystoma mexicanum) has an intrinsic ability to regenerate limbs and organs throughout life, it is an advantageous animal model for studying the physiological and molecular mechanisms of regeneration. Compared to zebrafish, a well‐established regenerative animal model of diabetes, the size of the axolotl (up to 150 g) is fit for pre‐clinical imaging systems and serial blood samples throughout an experiment without causing any detrimental effects.

We hypothesize that the axolotl has the ability to regenerate β cells, giving it the potential of becoming a new regenerative animal model of diabetes. To generate a diabetes‐like axolotl, streptozotocin, a cytotoxic glucose analogue targeting β cells specifically, was injected intraperitoneally. Initially, a protocol inspired by studies of zebrafish were used, in which 0.35 mg/g body mass was given at day 0, 1, 2, 11, and 18 to group 1 (n=12). However, this dosage caused high mortality, and the experiment was terminated prematurely. Therefore, three new dosage regimes inspired by rodent studies were investigated. For group 2 (n=3), animals were treated with 0.05 mg/g body mass at 5 consecutive days, group 3 (n=3) was given 0.05 mg/g body mass at day 0, 2, 4, 12, and 19, and group 4 (n=3) was given a single dose of 0.2 mg/g body mass at day 0. Disease development was detected by glucose tolerance tests and will further be investigated with immunofluorescence. Potential off target effects of streptozotocin will be characterized by examining changes in red blood cell morphology and the histology of liver and kidney.

We found that the treatment given to group 2 and 3 resulted in diabetes‐like symptoms when compared to a control group. Group 3 resulted in low‐grade hyperglycemia, whereas the 5 consecutive injections given to group 2 caused moderate hyperglycemia.

Our results demonstrate that the axolotl is able to develop diabetes‐like symptoms by streptozotocin treatment. We anticipate that further adjustments to the dosage regime given to group 2 will result in severe hyperglycemia and thus model a diabetic axolotl, and that this animal model will be able to regenerate the lost β cells. Thus, this animal model will be useful for studying the cellular source of the regenerated cells and the molecular mechanisms of regeneration.
Quantifying movement in ultrasound imagery of the hepatic‐piston pulmonary apparatus in the American alligator (Alligator mississippiensis)
Authors: Grand Pre Clinton A. , Thielicke William , Hedrick Brandon P. , Elsey Ruth M. , Schachner Emma
Published: May 2022

Crocodilians ventilate their lungs using a novel respiratory mechanism called the hepatic‐piston apparatus. Previous work presumes that the hepatic‐piston pump is facilitated by a smooth thoracodorsal ceiling. However, the sliding and displacement of the pleura has never been confirmed in living crocodilians. Using ultrasound we recorded images of the lung pleura, liver, and viscera freely sliding cranially and caudally in seven individuals (n=7) of Alligator mississippiensis. Currently, there are no marketed software packages available for use with sonography equipment to quantitatively measure movement in ultrasound videos. To accurately quantify pleural displacement, we used modified cross‐correlation functions from PIVlab, an open‐source program for particle image velocimetry (PIV). This technology is most often used to trace velocity of particles in fluids. Here we successfully implemented this technology to trace the coherent textures in ultrasound videos and measure lung and visceral displacement. We verified our results with screen captures that included scale bars of the most cranial and caudal positions of the liver during ventilation, a landmark clearly visible in ultrasound. In each animal, 16 second ultrasound videos were collected 1) in a calm, resting state with shallow breaths, and 2) inspiring a 5% CO2 fixed N2 gas to invoke deep breathing. Data were collected at an axillary position, a thoracic position, the pleural/hepatic boundary, and a post‐hepatic position. The ultrasound recordings show that in every instance, the pleura slides during ventilation. The total displacement varies based on two primary factors: 1) while breathing the 5% CO2 fixed N2 gas each individual took deeper breaths and the amount of pleura displacement increased and 2) there is a spatial gradient of displacement cranially to caudally, with minimal movement in the most cranial aspect of the lung and maximum movement caudally.