bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation of Magnetic Chitosan Beads as Carriers for Papain Immobilization
Authors: Ahmad Hassan , Garcia‐Rogers Juliet , Moreno Jacqueline
Published: May 2022

Papain, a well‐known cysteine protease, is predominantly found and extracted from papaya fruit. Its broad hydrolyzing activity and stability at different temperature and pH, make it a widely used enzyme in food, and pharmaceutical industries. For example, it is used in various food processing procedures and in the production of bioactive peptides. However, under extreme industrial conditions, the activity of this and other enzymes, are significantly decreased. This loss of enzyme activity could be overcome by immobilizing the enzymes on a solid support. The immobilization of enzymes also allows them to be reused. Chitosan is a non‐toxic, biodegradable polymer derived from the deacetylation of chitin, the second most abundant polysaccharide in nature. Chitosan offers multiple functional groups for binding enzymes and other molecules and has myriad enzyme immobilization applications on a wide variety of materials, including nanoparticles, nanocomposites, and nanofibers. In this study, we have prepared mechanically stable magnetic chitosan beads as carriers for immobilized papain. Chitosan beads were prepared using a phase inversion technique. Chitosan (2.0 gm) was dissolved in 50mL diluted acetic acid and mixed thoroughly with 0.3M FeCl3 solution. The homogenous mixture was placed in a syringe and extruded dropwise into a beaker with 0.5M sodium hydroxide with contestant stirring. The resulting beads were stirred for one hour and washed with water. The washed beads were incubated with 0.5% glutaraldehyde solution for 10 hours with constant shaking. This step was carried out to stabilize the beads by crosslinking the chitosan beads while also creating additional free aldehyde groups for papain binding. Following washing, the beads were immersed in 20mL of 10% ammonium hydroxide and 0.2M FeCl2 solution and stirred for 2 hours to create magnetic chitosan beads. The beads were separated using an external magnet and washed thoroughly. The beads were stored in 50mM phosphate buffer (pH 7.5). For immobilization of papain, the beads were incubated at room temperature in 0.1% papain solution for 20 hours with constant shaking. The immobilized papain was washed and stored in buffer. The enzyme activity of free and immobilized papain was determined by measuring the acid soluble peptides generated by hydrolyzed casein. The thermal stability studies shows that the immobilized papain retained 93% activity after 60 minutes incubation at 80oC while the soluble enzyme retained only 40% of its initial activity. There was no loss of immobilized papain activity after six weeks of storage at 4oC. In addition, a significant amount of immobilized papain activity is retained after multiple usage. These results show that attachment of papain on the magnetic chitosan beads could broaden its application in industrial use. Moreover, immobilization on magnetic chitosan beads can impede losses in activity of other industrially important enzymes.
Modulatory role of 17α‐estradiol (non‐feminizing estrogen) on 6‐OHDA‐induced Parkinsonism in ovariectomized rats
Authors: Medhi Bikash , Bansal Seema , Sharma Amit R.
Published: May 2022

Background

Epidemiological studies have reported that the incidence of Parkinson’s disease (PD) is 1.5 times more in men as compared to women. It suggests that gonadal hormones influence the development and progression of PD. Estrogen replacement therapies have a protective effect against dopaminergic toxins and can be protective in Parkinsonism. However, there are multiple unresolved issues regarding the safety of ERT. Thus, there is a essential need to find alternatives that would avoid these health risks.

Objectives

Thus main objective of study was to assess effect of 17α‐estradiol, a non‐feminizing estrogen, in attenuating PD employing an animal model of surgical menopause.

Methodology

17α‐estradiol and 17β‐estradiol was injected 10 µg/kg/s.c. for 14 days in ovariectomized 6‐OHDA rats. After 14 days of drug administration behavioral parameters of Parkinsonism (sensorimotor activity, motor coordination, and locomotor activity) were performed. These behavioral parameters were further correlated with various biochemical and molecular markers in postmenopausal Parkinsonism. Uterine weight and serum estradiol levels were also assessed to determine estrogenic effect of both the drugs peripherally. Effect of both drugs on expression of ER‐α and ER‐β in striatum was also evaluated. Further the differential effect of 17α‐estradiol and 17β‐estradiol was evaluated in postmenopausal Parkinsonism.

Results

17α‐estradiol and 17β‐estradiol equally modulated behavioral changes and attenuated rise in oxidative stress markers, inflammatory markers, improving neurotrophic factors and modulation of striatal neuron morphology. 17β‐estradiol treatment restored the uterine weight and serum estradiol levels towards normal, however 17α‐estradiol did not show any effect on uterine horns and serum estradiol levels. No effect of 17α‐estradiol was observed on expression of ER‐α and ER‐β in the striatum, indicates receptor independent effect of 17α‐estradiol

Conclusion

Both 17α‐estradiol and 17β‐estradiol are equally effective in Parkinsonism, however, 17α‐estradiol may prove to be safer alternative of 17β‐estradiol in Parkinsonism due to lack of feminizing effect.
Expression of Micrornas Mir‐126, Mir‐133b and Mir‐221 Associated With Apoptosis in Rats Submitted to Focal Cerebral Ischemia and Physical Exercise
Authors: Nunes Maria Julia , Figueiredo Rafael Z. , Tirapelli Vitor C. , e Silva Stevanato Ana Laura R. , Filho Sebastião de Campos Reis , Cirino Mucio Luiz d. A. , da Cunha Tirapelli Daniela P. , Tirapelli Luis Fernando
Published: May 2022

INTRODUCTION

Stroke is one of the main causes of death and disability in Brazil and worldwide. The great impact on the quality of life of the population and on the health system justifies that we seek relevant alternatives to reduce the incidence and improve the treatment and recovery of patients affected by this disease. Physical exercise appears as an important tool in this scenario, being already pointed out as a possible therapeutic approach for the prevention of non‐contagious chronic diseases. Studies have shown that physical exercise when used in a systematic and organized way produces positive effects on the individual's quality of life and health. In this context, biomarkers such as miRNAs that respond to physical exercise and are directly related to several epigenetic mechanisms appear. Therefore, explaining the molecular mechanisms involved during physical exercise will lead to a better understanding of each stimulus and the dose to be used to better respond to each situation, thus being a promising approach for the evolution of prescription and control of training and processes recovery from various diseases, including stroke.

MATERIALS AND METHODS

Forty‐eight Wistar rats were used, divided into four experimental groups: control group, ischemia group, physical exercise group and exercise + ischemia group. Real‐time PCR methodology was used to analyze the expression of miRNAs: miR‐126, miR‐133b and miR‐221.

RESULTS AND CONCLUSION

In our study we observed significant difference in miR‐221 expression between the control group and the other groups. However, microRNAs: miR‐126 and miR‐133b do not show significant differences in expression between groups.
Effects of Abolishing Whi2 on Nitrogen Catabolite Repression‐Sensitive GATA‐Factor Localization and Protein Production
Authors: Tate Jennifer J. , Marsikova Jana , Vachova Libuse , Palkova Zdena , Cooper Terrance G.
Published: May 2022

Gln3 and Gat1 are the transcriptional activators of nitrogen catabolite repression‐ (NCR‐) sensitive gene expression in Saccharomyces cerevisiae. In nitrogen replete medium, these activators are sequestered in the cytoplasm. As environmental nitrogen supplies dwindle, Gln3 and Gat1 enter the nucleus. There, they activate expression of the autophagy PI3‐kinase subunit gene ATG14 and the genes required to transport and catabolize whatever environmental nitrogen sources are still present. Two global protein kinase regulators, TorC1 and Gcn2, are major contributors to NCR‐sensitive regulation. Active TorC1, via Tor Associated Protein Tap42, binds to and inhibits two protein phosphatases, Sit4 and PP2A, required for nuclear Gln3 and Gat1 entry. When TorC1 is down‐regulated by limiting nitrogen or rapamycin treatment, the phosphatase‐Tap42 complexes are released and dephosphorylate Gln3 and Gat1, which can then enter the nucleus and mediate NCR‐sensitive transcription. Recently, a new regulator of TorC1 activity has been reported, Whi2 (Chen et al., 2018). The Whi2‐Psr1/2 complex down‐regulates TorC1 activity when primarily leucine concentrations are low. One of the reporters used to characterize Whi2 actions was an NCR‐sensitive DAL80‐GFP promoter fusion. We were puzzled by the results obtained with this reporter because Dal80‐GFP protein was detectable when cultures were grown in synthetic complete (SC) medium. We, a priori, expected GFP production to be undetectable in such nitrogen‐rich medium, because DAL80 expression is highly NCR‐sensitive. Therefore, we investigated the source of the GFP production and the NCR‐sensitive proteomes of wild type and whi2Δ cells. In the SC medium, Gln3 was efficiently sequestered in the cytoplasm and NCR‐sensitive DAL80 expression was only minimally present in either wild type or whi2Δ cells. The small amount of DAL80 expression observed could be attributed to Gat1‐mediated activation since this activator is more resistant to NCR‐sensitivity and hence is partially nuclear in nitrogen‐rich medium. Overall, however, proteins encoded by 109 of the combined 125 NCR‐sensitive genes, putatively NCR‐sensitive genes or genes containing GATA elements (the binding sites for Gln3 and Gat1) in their promoters were unaffected or completely absent when cells were cultured in the rich SC medium. The 16 proteins that did respond in the Whi2 experiments were largely those associated with amino acid transport and metabolism as might be expected from the high amino acid content of SC medium. We conclude that while Whi2 can be convincingly shown to down‐regulate TorC1 activity, it is not a significant regulator of NCR‐sensitive gene expression. These observations raise the important question of how a major downstream target of TorC1 remains immune to its down‐regulation by Whi2.
Osteological Correlates of Fascicle Length and Gape in Carnivora
Authors: Deutsch Ashley R. , Martens Lara L. , Hartstone‐Rose Adam
Published: May 2022

Previously, our lab has studied the scaling and dietary correlates of masticatory muscle fiber architecture separately in felids, musteloids, canids, and ursids as well as across the carnivoran order as a whole. In some of these lineages, fascicle lengths (FLs; a correlate of gape) scale with relative diet size, while across the order and divisions thereof, correlates of bite force (muscle mass and physiological cross‐sectional area; PCSA) predominantly scale with body size. In a subsequent recent analysis of 40 taxa spanning eight families from this broader sample, we found that volumetric estimators (e.g., the commonly used “dry‐skull” method described by Thomason) can be used to closely estimate muscle mass and PCSA from osteological remains alone. In the current investigation, we are using the same sample to evaluate osteological correlates of dissection‐based FL measurements. In a preliminary analysis of 14 musteloid taxa representing mustelids, mephitids, and procyonids, chosen because musteloid FL correlated best with diet size, we measured 13 origin to insertion distances to compare to the FLs of the masticatory adductors of these taxa. Raw individual and combined osteological distances were then regressed against FL for each individual muscle as well as average adductor FL using RMA regression. Residuals of same variables relative to body mass and cranial size were also compared. The most highly correlated raw osteological measurement for each muscle resulted in r2 values of 0.92‐0.65 for the best correlate of each adductor individually and their combination. However, correlations dropped substantially (r2=0.69‐0.29 for temporalis, masseter, and all adductors combined) when body size was accounted for, and the medial pterygoid osteological FL proxies accounted for almost none of the variation in measured FL (r2=0.08 for its highest proxy). Although the temporalis is the largest mandibular adductor, the best osteological proxies for relative FL were found for the masseter muscles (r2=0.69 vs. r2=0.29 for temporalis) – perhaps because the temporalis origin is affected by the neuro‐protective and sensory functions of the cranium, while the origin to insertion region of the masseter is almost entirely influenced by masticatory function. Combining these findings with measures of jaw leverage, it is possible to reconstruct not only FL in fossil musteloids but also gape – a method that may give new insight into relative diet sizes consumed by these taxa. Expanding our methods across the other members of the order will allow for the theoretical ex vivoapproximation of gape in modern taxa for which in vivo data have not been systematically collected and also myological approaches to gape estimation in extinct taxa like Smilodon and other sabertooths.
Characterization of Pulmonary Fibroblast Response to Aspergillosis fumigatus Exposure and Clinical Implications on the Development of Invasive Aspergillosis
Authors: Lowe Elliot K. , Jones Shannon , Guirao Abad Jose P. , Ivey Malina , Alam Perwez , Askew David S. , Kanisicak Onur
Published: May 2022

Aspergillosis fumigatus(AF) is a ubiquitous environmental mold that spreads by producing airborne conidia (spores). Playing a crucial role in the recycling of environmental carbon and nitrogen, A. fumigatus is inhaled abundantly by animals and humans alike. Unfortunately, it is also responsible for one of the worst life‐threatening pneumonias known as invasive aspergillosis with a mortality rate above 30%, even with treatment. Healthy individuals that are immunocompetent can quickly clear inhaled AF spores before their germination via immune cells. However, in immunocompromised individuals the inability to clear the fungus allows the spores to germinate releasing a plethora of proteolytic enzymes as they infiltrate the tissue resulting in severe damage to the lung. Recent studies have shown that the proper response of the immune system hinges upon complex cellular cross‐talk between resident interstitial cells and circulating immune cells. Tissue resident fibroblasts, which are responsible for the production and maintenance of the extracellular matrix (ECM), have been recently suggested to play a pivotal role in regulating immune response. We hypothesize that A. fumigatus‐induced damage to the lungs would result in activation of pulmonary fibroblasts (PFs) and aid in protective inflammatory response. Utilizing our recently developed in vivo cre‐loxP lineage tracing mouse models we have interrogated and manipulated all activated lung fibroblasts during AF infection, clearing, and reinfection. Data from our lab shows that PFs can be activated following acute inhalation of AF spores even when they are cleared from the lungs of immunocompetent mice before pathological tissue damage occurs. Furthermore, repeated AF infections which are pathologically asymptomatic result in the accumulation of activated PFs. Here we show for the first time that fibroblast activation occurs in both transient and chronic pulmonary infections revealing a role in the immunomodulatory response and subsequent development of pulmonary disease.
Biochemical characterization of a new 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) from Methanocaldococcus jannaschii
Authors: Lewis Benjamin T. , Allen Kylie , Gendron Aleksei , Stone Spenser
Published: May 2022

Methanogenic archaea are strict anaerobes that produce methane as a byproduct of their essential energy metabolism known as methanogenesis. Tetrahydromethanopterin (H4MPT), a specialized cofactor similar to tetrahydrofolate (H4F), is a C1carrier required for the methanogenesis pathway. The biosynthesis of H4MPT utilizes a similar series of reactions used in H4F biosynthesis, but they are catalyzed by nonorthologous enzymes. Here, we report the recombinant expression, purification, and enzymatic properties of MptE (MJ1634), a novel 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) identified to catalyze the final step in the biosynthesis of the pterin portion in H4MPT in Methancaldococcus jannaschii by catalyzing the transfer of a pyrophosphate from ATP to the substrate dihydro‐6‐hydroxymethylpterin (H2HMP). This novel HPPK shows a distinctive sequence from the bacterial counterpart; instead, it belongs to the superfamily of thiamine pyrophosphokinase (TPK) catalytic domain. The gene encoding MptE from M. jannaschii was overexpressed in Escherichia coli, and the his‐tagged recombinant enzyme was purified by metal‐affinity chromatography. After purification, initial enzyme assays were performed and analyzed via HPLC, which revealed a similar catalytic efficiency to bacterial HPPKs. Site‐directed mutagenesis studies coupled with structural analyses revealed that Asp106 and Asp171 are essential for Mg2+ coordination, His129 and Phe169 play important roles in H2HMP binding, and Lys217 is important for transition state stabilization. These residues were selected because they are strictly conserved in the archaeal protein family COG1634 or thiamine pyrophosphokinase (TPK), whose active site resembles those in the COG1634 family. Currently, kinetic studies are underway for additional variants to further identify key residues of structural and catalytic importance.
Chronic AICAR treatment enhances anabolic signaling in sarcopenic skeletal muscle
Authors: Smith Andrew H. , Brodowski Kole D. , Wilcox Shalene H. , Darrington Tyler , Thomson David M.
Published: May 2022

Many fields of study have grappled with the challenge of decelerating the effects of aging. Physical exercise plays a vital role in attenuating the impact of the aging process. While many exercise‐induced chemical signaling pathways are not fully understood, AMP‐activated protein kinase mediates many beneficial adaptations to exercise training. AMPK is activated by the accumulation of AMP (and ADP) during muscle contraction‐induced ATP breakdown. The prodrug AICAR mimics AMP accumulation and leads to AMPK activation by increasing the concentration of the AMP analog, ZMP. The primary purpose of the present study is to determine the effect of chronic (31 days) AICAR treatment on muscle anabolic signaling in old (24 mo.) mice. AICAR improved treadmill running performance and attenuated muscle atrophy in the aged mice. Surprisingly, given it's known acute effects on muscle anabolism, AICAR treatment increased the skeletal muscle concentrations of insulin‐like growth factor‐1 and apelin, both of which are known to activate anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Accordingly, ribosomal protein S6 phosphorylation was also elevated in AICAR‐treated muscles. Our results suggest that in addition to its other well‐established metabolic effects, chronic AICAR treatment may enhance anabolism in sarcopenic muscle and potentially in other muscle‐wasting conditions.
Effectiveness of X‐ray in Detecting the Fabella Sesamoid Bone
Authors: Alzahabi Obadah , Bauman Jay
Published: May 2022

The highly variable prevalence of the fabella, a sesamoid bone located posterior to the lateral condyle of the femur, invites further investigation. A recent meta‐analysis by Berthaume [J. Anat. 2019 Jul; 235(1): 67‐79. doi:10.1111/joa.12994] reports that the incidence rate of the fabella has increased over the past 150 years. Within the data aggregated by that study, there is also a significant difference in detecting the fabella based on the method of detection ‐ anatomical dissection (28.9% prevalence, N = 5723 knees) vs. X‐ray (15.3% prevalence, N = 13665 knees). We sought to investigate whether variation in the density of fabellae could explain both changes in its prevalence over time as well as the discrepancy between methodologies.

Our first aim was to directly compare the two methodologies, dissection and X‐ray, within the same set of knees on their determination of fabella prevalence. We hypothesized that fewer fabellae would be detected by X‐ray than dissection. Secondarily we are currently evaluating the hypothesis that some fabellae exist but are not sufficiently dense to be reliably detected via X‐ray.

Fifty human knees were isolated by transverse sections through the middle of the upper and lower legs. The knees were obtained from cadavers received through the Saint Louis University Gift Body Program of the Center for Anatomical Science and Education (CASE) with signed, informed consent from the donor. The CASE Gift Body Program abides by all the rules set forth by the Uniform Anatomical Gift Act. An X‐ray generator (AJEX Meditech) emitted rays at 70 kV, 2.0 mAs through the medial or lateral side of each knee into a photo‐sensitive plate. Images from the plate were digitized using TigerView software. Three raters assessed whether a fabella was present in the X‐ray images (e.g. Fig. 1) using inter‐rater agreement, while one individual subsequently performed anatomical dissection.

The prevalence rate via X‐ray was 12% (using a standard of 2/3 rater agreement). Fleiss’ kappa was 0.24 for X‐ray ratings, which represents a fair amount of interrater agreement. The prevalence rate based on anatomical dissection was 54%, which is significantly different from that of X‐ray (p < .0001, chi‐square statistic is 19.95).

These results confirm that X‐ray imaging underreports the prevalence of fabellae. This is to our knowledge the first study that directly compares X‐ray against dissection within the same subjects for sesamoid identification, which is important for diagnosing conditions such as fabella syndrome on presentation of posterolateral knee pain. The authors continue to analyze the fabellae obtained from anatomical dissection to determine why some sesamoids are not consistently visible under X‐ray.
Impact of Intermittent Hypoxia Exposure on Erythropoietin Levels in Older Individuals
Authors: Wojan Frank , Stray‐Gundersen Sten , Massoudian Sahar , Lalande Sophie
Published: May 2022

Few minutes of hypoxia exposure stabilizes hypoxia‐inducible factors, resulting in erythropoietin (EPO) gene transcription and production. A brief intermittent hypoxia exposure increased EPO levels in young healthy individuals, suggesting that a single session of intermittent hypoxia has the potential to increase oxygen‐carrying capacity. Thus, the objective of this study was to determine the effect of a single session of intermittent hypoxia on serum EPO levels and hemoglobin mass among older individuals. We hypothesized that a single session of intermittent hypoxia would raise serum EPO levels and lead to an increase in hemoglobin mass in older individuals. Seventeen participants (8 women, age: 54 ± 8 years, height: 177 ± 10 cm, weight: 76 ± 14 kg, BMI: 24 ± 4 kg/m2) were randomly assigned to an intermittent hypoxia group (IH, n=11) or an intermittent normoxia group (IN, n=6). Intermittent hypoxia consisted of eight 4‐minute cycles at a targeted arterial oxygen saturation of 80% interspersed with normoxic cycles to resaturation. Air was made hypoxic by titrating nitrogen into the breathing circuit. Intermittent normoxia consisted of the same protocol, but nitrogen was not added to the breathing circuit. Pulmonary gas exchange, arterial oxygen saturation, and hemodynamics were continuously measured throughout both protocols. EPO levels were measured before and 4.5 hours after the beginning of each protocol. Hemoglobin mass was assessed via carbon monoxide rebreathing the day before and seven following intermittent hypoxia or normoxia. Intermittent hypoxia lowered arterial oxygen saturation (­­98 ±­ 1 to 82 ± 3 %, p<0.01), which resulted in a lower fraction of inspired oxygen (20.8 ±­ 0.1 to 10.9 ± 1.0 %, p<0.01). There was no significant change in EPO levels in either condition (IH:10.4 ±­ 2.9 to 13.3 ± 4.2; IN: 5.6 ±­ 2.4 to 6.5 ± 2.9 mU/ml, main effect for time p=0.12). Similarly, there was no change in hemoglobin mass in response to both conditions (IH: 752 ±­ 189 to 754 ± 189; IN: 858 ± 177 to 879 ± 157 g, main effect for time p=0.87). Intermittent hypoxia did not affect mean arterial pressure (87 ± 15 to 88 ± 14 mmHg, p=0.18) or cardiac output (5.5 ± 1.5 to 5.7 ± 1.5 L/min, p=0.22), but increased heart rate (62 ± 9 to 68 ± 9 bpm, p<0.01). In conclusion, a single session of eight 4‐minute cycles of intermittent hypoxia did not increase serum EPO levels in older individuals.
Air Particulate Matter Significantly Alters Bacterial‐Derived Metabolites in APP/PS1 Mice
Authors: Mackos Amy R. , O'Piela Devin R. , Escobar Yael‐Natalie , Peterson Devin , Bernier Matthew , Somogyi Arpad , Wold Loren E.
Published: May 2022

Alterations to gastrointestinal bacterial communities have been linked to the exacerbation of numerous diseases, including Alzheimer’s Disease (AD), via changes to bacterial‐derived metabolites. Community changes can result from antibiotic use, dietary changes, psychosocial stress, and possibly air particulate matter exposure. Previous studies from our group demonstrated that a transgenic mouse model predisposed to create amyloid‐β fibers similar to AD, namely APP/PS1 mice, exposed to air particulate matter (PM) led to significant increases in hippocampal amyloid‐β1‐40 (Aβ) and Aβ1‐42, as well as astrocytosis, microgliosis, and proinflammatory cytokines in the temporal cortex as compared to PM‐exposed non‐carrier mice. Such findings indicate the presence of PM2.5‐induced neuroinflammation in APP/PS1 mice; however, the mechanism by which this occurs is unknown. Recent data demonstrates PM‐induced alterations to the gut microbiome, prompting us to test the hypothesis that disruptions of microbial‐derived metabolites are present in PM‐exposed APP/PS1 mice. To test this hypothesis, APP/PS1 and non‐carrier, littermate wild type (WT) mice were exposed to filtered air (FA) or PM for 5 days/week, 6 hours/day, for 3 months. The average daily PM exposure is 90.0 μg/m3, which is a 5‐fold concentration above the ambient mean daily PM2.5 concentration (18.8 ±11.4 μg/m3) at the study site in Columbus, OH. At the end of the 3‐month exposure, fecal samples were collected to assess bacterial byproducts, i.e. short chain fatty acids (SCFAs), by gas chromatography/mass spectrometry. Serum samples were collected to characterize additional metabolites including the bacterial metabolite trimethylamine (TMA) by liquid chromatography/mass spectrometry. Results demonstrated that SCFA butyric acid was significantly reduced by PM‐exposure in both APP/PS1 and WT animals as compared to their FA‐exposed counterparts (p<0.01) and propionic acid was significantly increased in FA‐exposed APP/PS1 mice as compared to their WT counterparts (p<0.0001). Serum metabolite analyses revealed distinct principal component patterns among groups, demonstrating unique metabolic profiles associated with exposure and genotype. One such metabolite, TMA, was significantly increased by PM‐exposure (p<0.05). Taken together, these findings indicate that airborne particulate matter exposure alters metabolic profiles in a transgenic mouse model of AD.
Characterization of an Unusual Metalloenzyme Containing an [FeFe] Hydrogenase and a Rubrerythrin Domain
Authors: Taylor Jesse D. , Silakov Alexey
Published: May 2022

[FeFe] hydrogenases are enzymes that catalyze the reversible reduction of protons to dihydrogen with staggering efficiency. As dihydrogen is a promising alternative energy carrier for renewable energy systems, these enzymatic systems are of interest to biotechnological applications. Members of the [FeFe] hydrogenase class are very diverse in the primary structure, especially in the metallocofactor composition, and their physiological roles. Understanding the structure‐function relationships in these enzymes provides an opportunity to engineer enzymes with uniquely modified characteristics. In this work, we aim to understand the interplay between structural elements and the phenotype of an unusual metalloprotein from Clostridium perfringens (CpHydR) containing two catalytic domains, [FeFe] hydrogenase and rubrerythrin, “fused” together into one protein. Although [FeFe] hydrogenases are typically biased toward H2 production, the postulated presence of rubrerythrin makes us hypothesize that CpHydR is biased toward H2 oxidation. Rubrerythrin is known to reduce H2O2 as a part of an oxidative stress response process; it requires the donation of two electrons and two protons, which can be supplied by the [FeFe] hydrogenase. Hydrogenases are notoriously oxygen‐sensitive enzymes inhibiting their industrial application. The postulated use of this metalloprotein in the oxidative stress response in Clostridium perfringens would suggest tolerance to O2. Here we present experiments confirming the presence of each subsystem and the postulated functionality. We utilize Fourier Transformed Infrared (FTIR) and Electron Paramagnetic Resonance (EPR) spectroscopies to elucidate the structure of the active cofactors. To understand the function of CpHydR, we employ electrochemical methods, including protein film voltammetry. Results indicate an intriguing deviation of the spectroscopic signatures of the [FeFe] hydrogenase subdomain from the commonly observed phenotypes of virtually any other known enzyme of this class. This divergence in the structural characteristics also coincides with an unexpected strong bias of this enzyme towards hydrogen oxidation.
Identifying Critical DO2 with Compensatory Reserve during Simulated Hemorrhage in Humans
Authors: Convertino Victor A.
Published: May 2022

Background

Hemorrhage is a leading cause of potentially‐survivable death in trauma. The basic principle for treatment of ongoing bleeding prior to hemorrhage control is that early detection and intervention will improve outcome. However, current monitoring technologies that rely on standard vital signs are unreliable in assessing the clinical status of trauma patients because of variability in the capacity of individuals to compensate for hypovolemia. A monitoring capability designed to measure changes in systemic oxygen delivery (DO2) would provide an earlier indicator of clinical status in patients with life‐threatening blood loss. Maintaining DO2 above the level associated with the onset of shock (i.e., DO2crit) until bleeding is controlled should be the aim of any hemorrhage intervention. But DO2crit has not been identified in humans. Over the past decade, measuring the physiological phenomenon of compensatory reserve (CRM) based on interrogation of arterial waveform morphology has provided a capability to assess the reduction in the capacity to compensate for ongoing progressive central hypovolemia. Based on non‐human primate hemorrhage experiments, we hypothesized that humans would demonstrate a linear relationship between CRM and DO2, allowing for the identification of a DO2crit in humans of ~5 to 6 ml O2·kg‐1·min‐1. We also hypothesized that individuals with high (HT) and low (LT) tolerance to hypovolemia (i.e., ‘good’ versus ‘poor’ compensators) would have similar CRM‐DO2 relationships and DO2crit.

Study Design and Methods

CRM and calculated DO2 were measured in 166 healthy subjects (97 males, 69 females) with a mean ± SD age of 23 ± 4 years, height of 171 ± 11 cm, and weight of 69 ± 11 kg. All subjects underwent progressive central hypovolemia induced by lower body negative pressure as a model of ongoing hemorrhage to a physiological state of hemodynamic decompensation. Subjects were classified as having either high tolerance (HT; N = 111) or low tolerance (LT; N = 55) to central hypovolemia based on whether they completed 5 minutes at ‐60 mmHg LBNP.

Results

HT and LT groups were matched for age, weight, BMI, and vital signs, DO2 and CRM at baseline. The CRM‐DO2 relationship was best fitted to a logarithmic model in HT subjects (R2 = 0.971) and a polynomial model in the LT group (R2 = 0.991). Average DO2crit for the entire subject cohort was estimated at 5.3 ml O2·Kg‐1·min‐1, but was ~14% lower in HT compared to LT subjects. The reduction in DO2 from 40% CRM to 20% CRM was 2‐fold greater in the LT compared to the HT group.

Conclusions

Average DO2crit in humans is 5.3 ml O2·Kg‐1·min‐1, but is ~14% lower in HT compared to LT subjects. The CRM‐DO2 relationship is curvilinear in humans, and different when comparing HT and LT individuals. A threshold for an emergent monitoring signal should be set to 40% CRM given that the decline in DO2 from 40% CRM to 20% CRM for LT subjects is located on the steepest part of the CRM‐DO2 relationship.
The Investigation on the Effect Asn123Gly mutation in the Enzyme Pyrroline‐5‐Carboxylate Reductase
Authors: Molla Wengelawit A. , Bruender Nathan A.
Published: May 2022

In this experiment, we are studying the enzyme pyrroline‐5‐carboxylate reductase from Sinorhizobium melloti. Bioinformatics proposed that the enzyme catalyzes the reduction of pyrroline‐5‐carboxylate to L‐proline using NAD(P)H, which is the final step in proline biosynthesis. The purpose of this experiment is to study the effect of the point mutation of asparagine 123 to glycine on the enzymatic activity of the enzyme. The mutation was generated using site‐directed mutagenesis to change the GGT codon to CCA in the proC gene. The mutation was confirmed via DNA sequencing and the gene was recombinantly expressed in Escherichia coli Rosetta 2 (DE3) cells for protein overproduction. The protein was purified using Ni‐NTA affinity chromatography to >90% purity (determined by SDS‐PAGE). The effect of the mutation on the function of the enzyme was determined using a continuous kinetic assay following the oxidation of the L‐proline analog L‐thiazolidine‐4‐carboxylate using NAD(P)+ and monitoring the formation of NAD(P)H at 340 nm on a UV‐visible spectrophotometer. Preliminary investigations show that the mutation results in a decreased kinetic activity. Current work is underway to identify the underlying structural reason why the Asn123Gly variant has decreased activity.
PHB Improves Metabolic Homeostasis in Obese C57BL6 Male Mice Fed a High Fat Diet
Authors: Hubbard Katelin N. , Puckett Dexter , Dowker Presley , Gill Nick , Bettaieb Ahmed
Published: May 2022

Pharmacological approaches to combat metabolic syndrome have been met with limited success. Given the critical role AMPK plays in metabolic homeostasis, targeting this protein as a potential treatment and prevention strategy for metabolic syndrome could be of clinical interest. Our current study investigated the effects of a well‐defined poly‐herbal blend (PHB) on high fat diet (HFD)‐induced body mass dysregulation and alterations in metabolic homeostasis and glycemic control in mice. Age‐matched mice were fed standard lab chow for six weeks, then switched to HFD supplemented with PHB. Preliminary data demonstrate that body weights of control and PHB‐treated mice were comparable in chow fed animals. However, mice fed HFD supplemented with PHB gained less weight compared to controls. Furthermore, PHB‐fed obese mice exhibited improved glucose tolerance and insulin sensitivity. Food intake of control and PHB‐treated mice were comparable on both diets. Consistent with the decreased body weight, PHB supplementation resulted in decreased fat mass. Mechanistic studies in adipocytes and muscle cells demonstrated that these beneficial effects were mediated, at least in part, through regulation of the AMPK signaling pathway. Taken together, our preliminary studies identify PHB as a natural and sustainable approach for the treatment and prevention of metabolic syndrome and warrant additional investigation.
Overexpression of human OGG1 improves skeletal muscle endurance in mice
Authors: Blaze Bhavya , Marfo Emmanuel , Sampath Harini
Published: May 2022

8‐oxoguanine glycosylase 1 (OGG1) is a DNA glycosylase that catalyzes the excision of oxidized guanines as part of the base excision repair (BER) DNA repair pathway. While OGG1 has been studied extensively for its role in tumor prevention and neurodegeneration, recent studies have indicated a novel role for this enzyme in maintaining metabolic health. In particular, deletion of OGG1 increased propensity to diet‐ and age‐induced obesity, as well as impaired skeletal muscle dysfunction. In contrast, overexpression of human OGG1 (hOGG1) significantly protected mice from diet‐induced obesity and related metabolic sequelae. Unbiased transcriptomics studies using mice constitutively overexpression OGG1 (Ogg1‐transgenic ‐Ogg1Tg) revealed significant transcriptional changes in skeletal muscle of these mice. Among the most highly induced genes, expression of the myokine fibroblast growth factor‐21(FGF21) was elevated by 9.9 fold in RNA‐Seq analyses. qPCR analysis further confirmed these changes in skeletal muscle without any alterations in the hepatic or cardiac expression of Fgf21. Commensurate with gene expression changes, plasma FGF21 was elevated by 11.2 fold in Ogg1Tg mice relative to WT controls. Conversely, Fgf21 gene expression in skeletal muscle and plasma FGF21 levels were decreased by 25 and 29% respectively in Ogg1‐/‐ mice. Consistent with increased FGF21 activity, phosphorylation of AMPK and its target acetyl CoA carboxylase (ACC), and Ser/Thr phosphorylation of Akt were significantly increased in Ogg1Tg muscle. FGF21 is known to increase expression of PGC‐1α protein. Consistently, Ogg1Tg mice had a 1.6 fold increase in PGC‐1α protein. Given these alterations, we asked if markers of muscle health such as endurance would be altered in Ogg1Tg mice. Interestingly, Ogg1Tg mice displayed a 3.4 fold increase in running capacity, consistent with improved skeletal muscle health in these animals. This improvement in exercise capacity was evident in both young and aged mice and have important implications to the management of age‐related declines in muscle health and DNA repair capacity.
Erythropoietin Produces a Dual Effect on Carotid Body Chemoreception in Male Rats
Authors: Arias‐Reyes Christian , Laouafa Sofién , Zubieta‐DeUrioste Natalia , Joseph Vincent , Bairam Aida , Schneider‐Gasser Edith M. , Soliz Jorge
Published: May 2022

Erythropoietin (EPO) regulates respiration under conditions of normoxia and hypoxia through interaction with the respiratory centers of the brainstem. Here we investigate the dose‐dependent impact of EPO in the CB response to hypoxia and hypercapnia. We show, in isolated “en bloc” carotid body (CB) preparations containing the carotid sinus nerve (CSN) from adult male Sprague Dawley rats, that EPO acts as a stimulator of CSN activity in response to hypoxia at concentrations below 0.5 IU/ml. Under hypercapnic conditions, EPO did not influence the CSN response. EPO concentrations above 0.5 IU/ml decreased the response of the CSN to both hypoxia and hypercapnia, reaching complete inhibition at 2 IU/ml. The inhibitory action of high‐dose EPO on the CSN activity might result from an increase in nitric oxide (NO) production. Accordingly, CB preparations were incubated with 2 IU/ml EPO and the unspecific NO synthase inhibitor (L‐NAME), or the neuronal‐specific NO synthase inhibitor (7NI). Both NO inhibitors fully restored the CSN activity in response to hypoxia and hypercapnia in presence of EPO. Our results show that EPO activates the CB response to hypoxia when its concentration does not exceed the threshold at which NO inhibitors masks EPO’s action.
Targeted Covalent Inhibition of Small CTD Phosphatase‐1 to Promote the Degradation of REST Transcription Factor in Human Cells
Authors: Zhang Yan , Medellin Brenda , Yang Wanjie , Siegel Dio
Published: May 2022

The repressor element‐1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. High level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small molecule inhibitors, the inhibition of a regulatory protein, Small CTD Phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,β‐unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time‐ and dose‐dependent manner with an EC50 ~1.5 µM, reducing REST protein levels and activating specific REST‐suppressed genes. These compounds represent a promising line of small molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.
Cucurbitacin B and Evodiamine imparts anti‐neoplastic effects against Glioblastoma Multiforme via PI3‐K/AKT signaling pathway
Authors: Mehta Vrutti V. , Benzeroual Kenza E.
Published: May 2022

Glioblastoma Multiforme (GBM) is a highly malignant and aggressive brain tumor. Patients’ survival is around 6 to 12 months even with chemotherapy and surgery. Temozolomide is a first line chemotherapeutic treatment prescribed along with surgery but due to the high resistance to the drug and a low patient survival rate, new drugs and drug combinations must continue to be investigated and developed. Evodiamine, a marketed supplement, and Cucurbitacin B, a compound obtained from natural plants, have been identified as promising candidates to treat various neoplasms but has not been well studied in GBM, and especially in combination. The present study investigated the anti‐proliferating effects of Evodiamine (Evo) and Cucurbitacin‐B (CuB) individually and in combination in U‐87MG cell line (Human Primary Glioblastoma cell line) and elucidated probable mechanisms of action via PI3K/AKT pathway in GBM. Results demonstrated that both CuB and Evo individually decreased the U‐87MG cell viability in a dose dependent manner. The effect was pronounced with drug combination. CuB and Evo imparted their effect via the PI3K/P‐AKT signaling pathway. In addition, Cells treated with CuB and Evo showed a decrease in Bcl2, an anti‐apoptotic factor, and an increase in Bax, a pro‐apoptotic factor, expression; a hallmark of induction of apoptosis. The increased expression of Cleaved Caspase‐3 and p‐c Jun have also been observed. Overall, Cucurbitacin and evodiamine individually and especially in combination proved to be potential anti‐neoplastic agents in U‐87MG cells and may be considered as potential adjuvant therapies for GBM.
Detecting Alterations in Fractal Fibrin Patterns using Light Microscopy
Authors: Skinner Kristen , Cordner Ryan
Published: May 2022

Fibrin is produced from the polymerization of fibrinogen at the completion of the coagulation cascade. Alterations in the structure and pattern of fibrin in plasma clots have been associated with various disease states such as pulmonary embolism, myocardial infarction, and hemophilia. Assessing these structural changes often requires confocal microscopy with fluorescently labeled fibrinogen or electron microscopy. Here, we demonstrate a novel method whereby pathophysiological alterations in fibrin patterns can be detected using light microscopy. Images of plasma clots from the partial thromboplastin time test were analyzed for alterations in fractal patterns using The Workflow of Matrix Biology Informatics. Factor VIII and Factor IX deficiencies showed significant changes in lacunarity, branchpoints, and total pattern area. We also detected significant changes in fibrin patterns altered by hyperglycemia, a known risk factor for thrombosis and embolisms. This work has direct clinical relevance as it utilizes readily available technology that can be applied in the clinical laboratory to screen patient samples for fibrin alterations when clotting time tests are prolonged. This diagnostic information could be utilized in the identification of various diseases that affect blood clot formation.
How Do Bacteria Recognize Their Environment? The Role of a Histidine Kinase in Initiating a Biofilm Program
Authors: Wacker Sarah , Figueroa Khaitlyn
Published: May 2022

Many bacteria form complex multicellular communities known as biofilms. While biofilms are a common cause of human infections, they can also have productive roles in ecology and agricultural settings. In contrast to the importance of these communities, less is known about how bacteria interpret and respond to environmental cues to form a biofilm. To help understand how bacteria recognize chemical signals from their environment, we studied the soil‐dwelling bacteria Bacillus subtilis, which is known to form beneficial biofilms on plant roots. We find extracts from many plant samples can stimulate B. subtilis biofilm formation. One protein that is likely important for the recognition of plant signals is a histidine kinase called KinD. KinD is known to have a role in biofilm formation and also to bind the molecule pyruvate. We find there are varying pyruvate concentrations in our different biofilm‐stimulating plant samples. Using transcriptional reporters of biofilm genes, we show that the binding of pyruvate to KinD can initiate the primary biofilm pathway of B. subtilis. However, the addition of pyruvate to bacterial cultures is not sufficient to trigger a biofilm phenotype. This research suggests that the binding of pyruvate to KinD is one of several signals that initiate the formation of a robust biofilm.
Characterizing the Role of the Neuroregulatory Protein Piccolo in Secretion of Insulin from Beta Pancreatic Islet Cells
Authors: Bush Gerald F. , Fenster Steven D.
Published: May 2022

Secretion of insulin from pancreatic beta‐cells is required for glucose homeostasis; however, the molecular mechanisms of insulin secretion have not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through a calcium (Ca2+)‐mediated Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified in neurons as active zone proteins that regulate neurotransmitter secretion. It has been demonstrated that Piccolo is downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). We hypothesize that Piccolo plays a key role in regulated insulin secretion and further study of Piccolo in pancreatic islets cells may provide valuable insights into the etiology of T2D. The C‐terminus of Piccolo contains a Ca2+‐binding C2A domain and we hypothesize that in pancreatic beta‐cells, Piccolo may regulate insulin secretion by serving as a Ca2+‐sensor. Alternative splicing within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (e.g., short C2A) or a variant that retains this exon (e.g., long C2A). Due to this splicing event, the short C2A domain lacks 9 amino acids resulting in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the Piccolo splice variants in Ca2+‐mediated insulin secretion using a rat pancreatic beta‐cell line (INS‐832/13). We demonstrate that INS‐832/13 cells express both variants containing either the short or long C2A domains. We also demonstrate that these cells express an additional splice variant called Piccolino previously thought to be only expressed in neurons associated with the eye and ear. To further assess the role of Piccolo in regulating insulin secretion, we plan to use a gene knockdown approach employing multiple shRNAs expressed in lentivirus that target Piccolo RNA sequences. To assess the effect of knockdown of Piccolo, we will stimulate ß pancreatic islet cells with increasing glucose concentrations to quantify changes in insulin secretion.