bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxygen Dose: A new perspective
Authors: MacLaughlin Kent , Lamers Jacob J. , Braun Rudolf , Eldridge Marlowe W.
Published: May 2022

Introduction

This study followed up on our findings, published in 2019, of stem progenitor cell mobilization following intermittent low level hyperoxia (42%) in an animal model. This most recent study in human participants used a never before tested small dose of intermittent hyperbaric air that has been used for many years as a placebo control in hyperbaric oxygen (HBOT) research. Because of the reports of a placebo response using this small dose in HBOT research performed by the Department of Defense and conflicting reports disputing the placebo response, coupled with the lack of evidence in the literature that this dose had ever been tested, we questioned if this a placebo or a dose response.

In this research human participants breathed room air in a portable hyperbaric chamber (mountain sickness chamber) (Vitaeris 320 Oxyhealth LLC) at 1.3 atmospheres absolute (ATA), daily for 10 days. This hyperbaric hyperoxia level is similar to 28% oxygen concentration in normobaria.

We hypothesized that the low dose of intermittent hyperoxia would mobilize stem cells in humans similar to our animal study.

Methods

Eight adult subjects were exposed to 1.3 atmospheres absolute (ATA) in a mountain sickness bag daily for 60‐minutes, 10‐times (M‐F) over a 12‐day period. Venous blood draws were taken at four time points. (1) Immediately preceding the first treatment (control), (2) Immediately following the first treatment, (3) Prior to the 10th treatment, and (4) 72‐hours after final treatment. We analyzed blood samples using flow cytometry for changes in the expression of surface markers CD45, CD34 and CD133 and performed a statistical analysis using a Friedman test followed by a paired Wilcoxon signed‐rank test.

Results

We found a nearly 2 fold increase in the expression of CD45dimCD34+CD133‐prior to the 10th treatment (9.8% to 18.9% = 194% increase) and a 3 fold increase 72 hours after the 10th treatment (9.8% to 29.6% = 303% increase). We also found CD45dimCD34‐133+ decreased by 85% between the control time point and prior to the 10th treatment (15% to 2.3% = 85% decrease) and further decreased to a decrease of 84%, 72 hours after the final treatment (15% to 2.0% = 87% decrease).

Conclusions

We conclude that breathing room air at 1.3 ATA daily for 60 minutes over a 2 week period has significant effects on the mobilization of stem progenitor cells and therefore is physiologically active. These findings suggest that 1.3 ATA hyperbaric air breathed daily is a dose treatment and not a placebo nor a sham. These data suggest a re‐evaluation of its use as a placebo control in scientific research is warranted.
Identification of Small Regulatory RNA Transcripts in Extracellular Vesicles from Lactic Acid Bacteria
Authors: Busby L. B. , Hoxha Klea , Oakes William C. , Ward Nicole M. , Pérez‐Alvarado Gabriela C. , Lee Brian M.
Published: May 2022

Extracellular Vesicles, also referred to as EVs, are spherical lipid membrane‐bound vesicles produced by both Gram positive and Gram negative bacteria. These vesicles are secreted into the extracellular space and play important roles in cellular and host communication, elimination of competitors, virulence, detoxification of environmental stress, and nutrient sensing. They are often packed with proteins, enzymes, lipids, nucleic acids and other biomolecules. Streptococcus thermophilus is a lactic acid bacteria (LAB), inhabiting the human digestive tract, that has been shown to produce EVs. The bacterial florae are known to influence the host immune system, metabolism, and neurological processes, but little is known about the biochemical pathways involved. Since EVs are involved in host communication, they may play a key role in affecting the biochemical processes of the host. To study the content and potential effects of EVs, we have grown different LAB strains under anaerobic conditions, including S. thermophilus, Lactobacillus acidophilus, and Lactobacillus bulgaricus. Our goal is to isolate EVs from these LAB strains and identify small regulatory RNA (sRNA) molecules that mediate communication and biochemical effects on the host systems. As part of this study, we have begun to identify sRNA genes in LAB strains that may mediate communication with other bacteria or the host. In S. thermophilus, we have focused on the AsdS sRNA transcript, that is 152 base pairs in length, and is involved in quorum sensing. This gene is conserved among other streptococcal species and in the human pathogen, S. pyogenes, a homologous sRNA, known as MarS, is associated with the regulation of virulence factors. Since S. thermophilus is a non‐pathogenic species, the AsdS gene cannot be involved in virulence, but based on functional bioinformatics predictions, it may be responsible for intraspecies communication, biofilm formation, and transport processes. Our current studies aim to identify additional sRNA transcripts that may be found in EVs and mediate effects on the host systems.
The Cold Receptor TRPM8 Mediates the Antioxidant and Cytoprotective Effects of Mild Hypothermia in Cerebral Endothelial Cells During Inflammation
Authors: Zhang Rong , Liu Jianxiong , Parfenova Helena
Published: May 2022

Cerebral vascular endothelium, the key component of the neurovascular unit, is most vulnerable to oxidative stress injury. Our previous reports in newborn pigs indicated that brain hypothermia prevents cerebral endothelial injury by oxidative stress caused by neonatal seizures and asphyxia. We used primary cerebral vascular endothelial cells (CVEC) from newborn pigs as an in vitro model of oxidative stress condition in the brain caused by inflammation. During normothermia (37˚C), CVEC respond to a pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) by excessive formation of reactive oxygen species (ROS) leading to cell death by apoptosis (caspase‐3 activation, DNA fragmentation, loss of cell‐cell contacts). Remarkably, mild hypothermia (24.5°C) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha. We tested the hypothesis that the cold sensing menthol receptor TRPM8 mediates the cytoprotective effects of mild hypothermia in cerebral endothelium. TRPM8 (mRNA and protein) is expressed in CVEC. The TRPM8 agonist icilin (5 µM) reduced ROS and prevented apoptosis in CVEC exposed to TNF‐alpha at 37˚C, thus mimicking the cytoprotective effects of mild cooling. The TRPM8 antagonist AMTB (20 µM) reversed the icilin‐mediated cytoprotective effects during inflammation. The cell surface biotinylation technique suggested that mild hypothermia and icilin (at 37˚C) promote a time‐dependent TRPM8 translocation from an intracellular compartment to the plasma membrane concomitantly with the receptor activation. We used the in vitro model of the blood‐brain barrier (BBB) to test the functional consequences of TRPM8‐mediated cytoprotection. Confluent monolayers of CVEC cultured on transwell inserts with semipermeable membranes were exposed to TNF‐alpha during normothermia and mild hypothermia conditions. The BBB properties were detected by transendothelial electrical resistance (TEER) and the permeability to 3 kDa fluorescent Dextran. During a 2‐6 h exposure to TNF‐alpha at 37°C, TEER was time‐dependently reduced, and the Dextran permeability was elevated indicating the BBB opening. Mild hypothermia (24.5°C) and icilin (at 37˚C) largely preserved the BBB properties during inflammatory conditions. Overall, our findings demonstrate that mild hypothermia, via a TRPM8‐mediated mechanism, reduces oxidative stress, promotes cell survival, and prevents BBB damage during inflammation. The TRPM8 targeting approaches may provide a promising therapeutic strategy for cerebrovascular disease caused by brain inflammation.
The Sulfiredoxin‐Peroxiredoxin axis promotes urethane‐induced lung adenocarcinoma through the regulation of the tumor microenvironment
Authors: Hao Yanning
Published: May 2022

Lung adenocarcinoma is the most common type of lung cancer and has a strong association with tobacco smoking. Smoking leads to the accumulation of reactive oxygen species (ROS) and oxidative stress damages to macromolecules, leading to abnormal cell growth & proliferation, transformation, changes in cell metabolism and a variety of other physiological functions. Accumulation of ROS only leads to increased expression of cellular antioxidants that contribute to tumorigenesis and cancer progression. Sulfiredoxin (Srx) is the only enzyme that reduces over‐oxidized forms of typical 2‐Cys Peroxiredoxins (Prxs). Compared to other Prx family members, Srx preferentially interacts with and has a higher binding affinity to Prx4. Our previously work demonstrated that the Srx‐Prx4 axis enhances the RAS/RAF‐MEK‐ERK signaling cascade to promote lung cancer cell proliferation and metastasis. However, the role of the Srx‐Prx axis in de novo lung tumorigenesis has not been explored.

In this study, Prx4‐/‐ mice and Srx‐/‐Prx4‐/‐ mice were generated and mouse lung carcinogenesis was induced by a well‐established urethane protocol. Briefly, wildtype and knockout mice at 8‐week of age were injected intraperitoneally with 1g/kg of urethane once per week for three successive weeks. Ten weeks after the administration of urethane, all mice were euthanized and examined for tumors. Our results indicate that both knockout mice have significantly reduced rates on tumor incidence, multiplicity, and size. Compared with tumors from wildtype mice, the rate of cell proliferation in tumors from either knockout mice is significantly decreased.

Macrophages are the most abundant immune cells identified in the tumor microenvironment of solid tumors and their presence correlates with reduced survival in most cancers. In addition to all other markers, we also compared the presence of two distinct states of polarized macrophages including the classically activated macrophage (M1) and the alternatively activated macrophage subsets (M2) in urethane‐induced tumors using specific surface marker staining. We found that there are significantly increased numbers of M1 cells in tumors of either Prx4‐/‐ or Srx‐/‐Prx4‐/‐ mice compared with those of wildtype mice. In contrast, there are significantly few numbers of M2 cells in tumors from either knockout mice.

It has been shown that M1 macrophages have immune‐stimulatory properties through the expression of a series of pro‐inflammatory cytokines, chemokines, and effector molecules, whereas M2 cells express a wide array of anti‐inflammatory molecules. Therefore, the difference in tumor‐associated macrophages caused by the loss of Prx4 or Srx and Prx4 may contribute to the reduced lung tumorigenesis observed in the mouse model. In addition, bioinformatic analysis shows that Prx4 is overexpressed in the majority of cancers, and the increased expression of Srx and Prx4 both show a strong correlation with poor prognosis in lung adenocarcinoma patients.
Activity‐based chemoproteomics reveals targets of phospholipase D‐derived phosphatidyl ethanol lipids
Authors: Yu Weizhi , Lin Zhi , Woo Christina , Baskin Jeremy
Published: May 2022

Alcohol consumption leads to formation of phosphatidylethanol (PEth) via the transphosphatidylation activity of phospholipase D (PLD) enzymes. Though this non‐natural phospholipid routinely serves as a biomarker of chronic alcoholism, its pathophysiological roles remain unknown. We use a minimalist diazirine alkyne alcohol as an ethanol surrogate to generate clickable, photoaffinity lipid reporters of PEth localization and lipid–protein interactions via PLD‐mediated transphosphatidylation. We use these tools to visualize phosphatidyl alcohols in a manner compatible with standard permeabilization and immunofluorescence methods. We also use click chemistry tagging, enrichment, and proteomics analysis to define the phosphatidyl alcohol interactome. Our analysis reveals an enrichment of putative interactors at various membrane locations, and we validate one such interaction with the single‐pass transmembrane protein basigin/CD147. Further, we identify binding sites of phosphatidyl alcohols to basigin/CD147 via both proteomics and targeted mutagenesis. This study provides a comprehensive view of the molecular interactions of phosphatidyl alcohols with the cellular proteome and points to future work to connect such interactions to potential pathophysiological roles of PEth.
An Unexpected Role for Mediator of ErbB2 Induced Cell Migration, MEMO1, During Amelogenesis
Authors: Van Otterloo Eric , Kiel Michaela , Wuebker Samantha
Published: May 2022

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel related disorders or more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we identified that Memo1 is expressed in ameloblasts and that conditional deletion of Memo1 from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal associated genes and a reduction in late stage ameloblast markers, relative to controls. Histology revealed that the molecular defects correlated with a disruption of the apical Tome’s process and the basolateral interacting, papillary layer, in Memo1 mutant ameloblasts. Finally, deletion of Memo1 from an oral epithelial ameloblast cell line, followed by cellular and molecular analyses, revealed altered cytoskeletal networks, relative to control cells. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide both in vivo and in vitro systems to further interrogate the relationship of cytoskeletal and amelogenesis‐related defects.
Calmodulin availability regulates basal autophagy
Authors: McConnaha Elizabeth , Giles Jennifer , Wauson Eric , Tran Quang‐Kim
Published: May 2022

Macroautophagy (hereafter, autophagy) is a multi‐step process through which cells degrade damaged or unutilized proteins or organelles (cargos) to maintain homeostasis. In the final step of autophagy, cargos are degraded by lysosomal enzymes, whose activities require an acidic environment. Intracellular Ca2+ has been shown to play diverse roles in autophagy. In many cases, Ca2+ signals are transduced into cellular activities by forming a complex with the ubiquitous Ca2+‐binding protein calmodulin (CaM), which then activates numerous target proteins, estimated to be over 300. However, CaM is not expressed sufficiently for all these targets. This generates a limiting condition in which CaM availability is competed for. Although several CaM‐binding proteins have been implicated in autophagy, the role of CaM availability in autophagy is unknown, especially in basal condition when there are no stimulated increases in intracellular Ca2+. Here we show that overexpressing CaM promotes autophagic flux in basal, unstimulated condition. Removal of amino acids triggers acute intracellular Ca2+ signals, and autophagic flux during this time frame is also significantly higher in CaM‐overexpressing cells compared to the wildtype counterparts. Inhibiting CaM using various CaM antagonists is associated with increases in lysosomal pH and accumulation of LC3‐II. Finally, buffering cellular free CaM using a high‐affinity CaM‐binding protein that can bind CaM at resting Ca2+ levels significantly increases basal lysosomal pH. These data indicate that CaM availability aids in maintaining optimal lysosomal pH, which contributes to the regulation of basal autophagy.
Therapeutic effects of L‐lysine in Dahl SS rats, a Model of Salt‐Induced Hypertension
Authors: Dissanayake Lashodya V. , Levchenko Vladislav , Palygin Oleg , Staruschenko Alexander
Published: May 2022

Our previous studies have suggested that dual treatment of glomerular filtration and albumin reabsorption might be an effective approach to treating salt‐sensitive (SS) hypertension. L‐lysine, an essential amino acid for humans, has been shown to inhibit tubular protein reabsorption with rapidly increasing urinary albumin excretion in healthy male participants. Our recent metabolomics study revealed that intrarenal levels of L‐Lysine are reduced in Dahl SS rats when animals are fed a high salt diet. In our initial experiments, we tested the effects of L‐lysine during the development of hypertension. We found that L‐lysine administration (17 mg/ml drinking water supplement) in SS rats restores albumin balance, triggers diuresis, and diminishes the development of SS hypertension. In our following experiments, we utilized a therapeutic approach to testing the hypothesis that L‐lysine could decrease the blood pressure of subjects with developed hypertension. The SS rats were initially kept on a 0.4% NaCl diet. When they reached 8‐weeks in age, rats were challenged with an 8% NaCl high salt diet (HS) for 10 days until they exceeded an average mean arterial pressure (MAP) of 140 mmHg (N=13; MAP was measured by implanted telemetry). Then, rats were randomly assigned to treatment and control groups. After another 11 days, all rats were euthanized and tissue was collected for further analyses. At the endpoint, the hypertensive L‐lysine treated rats had significantly lower MAP than hypertensive vehicle‐treated rats (145±5 mmHg vs. 176±8 mmHg). Interestingly, therapy with L‐lysine treatment did not result in a significant difference in diuresis or albuminuria. Excretion for all major electrolytes was comparable between the two groups except for Cl‐ (Cl‐/Creatinine for vehicle vs treated: 142±8 & 209±19). Increased excretion in Cl‐ is most likely due to the administration of L‐lysine as a chloride salt. Preliminary analysis using immunohistochemistry suggests less kidney damage in the treatment group compared to the vehicle group. Therefore, our data propose that L‐lysine, when used as a therapeutic, can effectively treat SS hypertension.
Effect of BAY‐117082, a NLRP3 inflammasome inhibitor, in a mouse model of nitroglycerin (NTG)‐induced migraine
Authors: Cuzzocrea Salvatore , Campolo Michela , Scuderi Sarah A. , Filippone Alessia , Lanza Marika , Casili Giovanna , Paterniti Irene , Esposito Emanuela
Published: May 2022

Migraine is a common brain disorder characterized by recurrent episodes of headache. It is a complex and multifactorial disease with a higher prevalence in females than in males. Several risk factors have been associated to migraine disease as genetic factors, sex and age. The pathophysiology of migraine is still unclear; however, it has been proven that NOD‐like receptor protein 3 (NLRP3) inflammasome pathway overactivation as well as p‐ERK/p‐CREB axis contribute to migraine pathogenesis. Therefore, the aim of this study was to investigate the effect of BAY‐117082, a NLRP3 inflammasome inhibitor and p‐ERK/p‐CREB modulator, in an in vivo model of nitroglycerin (NTG)‐induced migraine.

Migraine model was induced by NTG intraperitoneal administration at dose of 10 mg/kg diluted in 0.9% saline. Mice were treated intraperitoneally with BAY‐117082 at doses of 1 mg/kg, 5 mg/kg, and 10 mg/kg, 5 minutes after NTG injection. Mice were sacrificed 4 h following NTG injection; the whole brain with the rostral spinal cord was removed to perform several analysis.

Our results demonstrated that, following behavioral tests for pain and photophobia, BAY‐117082 treatment at doses of 5 mg/kg and 10 mg/kg reduced pain attacks induced by NTG more than BAY‐117082 at the dose of 1 mg/kg. Moreover, the treatment with BAY‐117082 at doses of 5 mg/kg and 10 mg/kg significantly reduced histological damage in the trigeminal nerve nucleus and significantly decreased inflammosome activation by reduction of NLRP3, ASC, IL‐1β and TNF‐α expression. Additionally, the treatment with BAY‐117082 at doses of 5 mg/kg and 10 mg/kg significantly modulated p‐ERK/p‐CREB axis activation through the reduction of p‐ERK, p‐AKT, p‐CREB and p‐PI3K expression.

Therefore, the obtained results offer new insight into the role of NLRP3 inflammasome pathway and p‐ERK/p‐CREB axis in migraine pathogenesis, suggesting that BAY‐117082 could be considered a novel therapeutic strategy to treat migraine.
Efficacy of a Patented Menthol‐Fortified Phytochemical Formulation in the Alleviation of Joint Pain and Inflammation in Human Subjects: A Clinical Investigation of HerboJoint
Authors: Bordoloi Binoy K.
Published: May 2022

Standardized botanical formulations have often yielded promising results in the treatment of debilitating joint pains such as those encountered in Osteoarthritis (OA) and Rheumatoid Arthritis (RA). We report a novel formulation of essentials oils made from the Generally Recognized as Safe (GRAS) family of plants widely used in Ayurveda namely Kattrna (CC‐Cymbopogon citratus) (3.0%), Sati (Hedychium spicatum) (1.0%) and Tumuru (ZA‐ Zanthoxylum alatum) oil (1.0%) along with Menthol (M‐ Mentha arvensis) (1.3%) in a non‐greasy cream base which can provide relief against joint pains arising out of arthritis. The formulation was designed as a topical cream for applying around the affected joint. Its purity was confirmed through HPLC and GC‐MS analysis by comparing with identifiable marker components. The formulation patented as HerboJoint was able to substantially bring down the levels of inflammatory cytokines TNF‐α, IL‐6 and IL‐1β in a Type II collagen induced mouse arthritis model. In human clinical trials, the formulation significantly decreased joint pain, joint swelling and joint stiffness arising out of Osteoarthritis and Rheumatoid Arthritis by 2.2, 2.7 and 3.6 times respectively and provided relief to about 90% of patients selected randomly. The corresponding TNF‐α levels in the sera of patients also decreased substantially thus indicating that the formulation was effective in management of both OA and RA. The present invention based on synergistic action of Essential Oils and Menthol is believed to provide a significant relief to these extremely painful and potentially incurable medical conditions.
A CURE for Cloning: Implementing a Modular Approach to Cloning Recombinant Antibodies in an Undergraduate Teaching Lab
Authors: Brooks Teresa , Brooks Cory
Published: May 2022

An undergraduate course‐based research experience (CURE) is a high impact practice which provides genuine research experiences to a large and diverse student population. We have implemented a six‐week research module involving the cloning and expression of therapeutic antibodies as the culmination of a senior level Biochemistry lab course. We have designed a cloning system that takes advantage of the modular domain structure of antibodies to facilitate changes to an antibody’s affinity, isotype, or allotype. Specifically, our system was developed for readily swapping the antigen‐binding N‐terminal sequences of different antibodies into a set of vectors encoding the conserved regions of the heavy and light chains, all in the background of a strong mammalian expression vector. Gene fragments corresponding to the constant regions of each immunoglobulin chain, as well as the antigen‐binding regions of unique antibodies were designed, and produced by gene synthesis. Various iterations of the module were tested over four different semesters with high levels of success in terms of student engagement and research goals. The resulting plasmid constructs are now being used by our research lab as part of the investigation of the nature of antibody recognition of cancer‐specific Mucin antigens. This project allowed our senior undergraduates to contribute to an active research program, while earning credit toward a required course. It exposed them to modern cloning methodologies, bioinformatics tools, cell culture techniques and participation in a local research conference. It is also a readily scalable project as only the variable regions of new antibodies of interest need be synthesized.
Global PER1 knockout Dahl Salt Sensitive rats show increased expression of renal Edn1 mRNA and Endothelin‐1 peptide
Authors: Juffre Alexandria , Costello Hannah , Crislip G. Ryan , Douma Lauren A. , Cheng Kit‐Yan “. , Bratanatawira Phillip , Zietara Adrian , Dissanayake Lashodya V. , Staruschenko Alexander , Gumz Michelle L.
Published: May 2022

Background

Previously, we showed that global knockout of the circadian clock protein PERIOD1 (PER1) in Dahl Salt Sensitive (SS) rats resulted in worsening of the SS hypertensive phenotype (1). PER1 KO rats also exhibited evidence of renal injury and increased plasma aldosterone levels after three weeks on a high salt diet (4% NaCl). Because we previously showed that PER1 negatively regulates the peptide hormone Endothelin‐1 (ET‐1) via transcriptional control of the ET‐1‐encoding gene Edn1, we wanted to investigate ET‐1 regulation in this novel rat model. Interestingly, Edn1 is also positively regulated by aldosterone through the mineralocorticoid receptor (MR).

Hypothesis

Given that ET‐1 signaling can be pro‐hypertensive and profibrotic in the kidney, we hypothesized that ET‐1 peptide levels in the kidney may be upregulated as part of the phenotype of the PER1 KO SS rats.

Methods

Protein and total RNA were isolated from the kidney samples of control and PER1 KO SS rats on normal diet (0.4% NaCl) or after three weeks on a high salt (4% NaCl) diet. Renal ET‐1 peptide and plasma aldosterone levels were measured by ELISA and quantitative PCR was performed to quantify the renal mRNA levels of ET‐1 and its receptors ETA and ETB, MR, and GR. Renal MR protein levels were measured by Western Blot and quantified by relative densitometry. Data were evaluated for genotype or diet differences using ANOVA.

Results

Renal ET‐1 peptide levels were significantly higher in PER1 KO rats on high salt diet when compared to control SS rats (p=0.0003), as were plasma aldosterone levels (p=0.0156). There was also a significant genotype difference in Edn1 mRNA levels, with increased levels in PER1 KO rats on a high salt diet (p=0.0389). In contrast, significant differences were not observed in the mRNA levels of ET‐1 receptors Ednra and Ednrb between genotypes or diets. Importantly, quantification of renal MR protein levels showed marked increase with genotype and treatment effects (genotype: p=0.0002, treatment: p=0.0133).

Conclusions

These data suggest that PER1 KO results in increased ET‐1 in the kidney, due to increased transcription of the Edn1 gene, with increased plasma aldosterone and renal MR protein levels. This apparent negative regulation of ET‐1 by PER1, coupled with increased MR and aldosterone levels, is likely to contribute to the hypertensive and renal injury phenotype of PER1 KO SS rats on a high salt diet. Together these data show an important role for PER1 and its target ET‐1 in salt‐sensitive hypertension and the accompanying renal injury.

Reference

1 Spires DR, Zietara A, Levchenko V, Gumz ML, Staruschenko A. Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat. FASEB J. 2020;34(S1):1.
Endothelin‐1 Receptor B (ETB) Deficient Rats Fed High Salt Diet Display Differential Mitochondria‐Related Gene Expression in the Kidney Cortex and Outer Medulla
Authors: Soto Miranda Brian , Biswal Sara N. , De Miguel Carmen
Published: May 2022

Levels of the vasoactive peptide endothelin‐1 (ET‐1) are persistently elevated in patients and animal models of salt‐sensitive hypertension. Our group recently reported that loss of ETB receptor function and the consequent overactivation of the ET‐1/ETA receptor axis are associated with exaggerated kidney damage, T cell inflammation and apoptosis under high salt conditions. Importantly, the molecular mechanisms by which ET‐1 leads to high salt‐induced renal damage remain unclear. These studies aimed to determine if the overactivation of the ET‐1/ETA axis alters the kidney expression of genes involved in mitochondrial biogenesis and function during high salt consumption. Male ETB deficient (ETB def) and transgenic (TG) control rats were placed on a high salt diet (HSD, 4.0% NaCl) for 3 weeks. At the end of this HSD period, 24h urine was collected and kidneys were separated in cortex and outer medulla. Urinary protein excretion was measured as a marker of kidney damage and the kidney expression of 84 mitochondria‐related genes was assessed by qRT‐PCR arrays. Compared to TG controls, high salt‐fed ETB def rats presented with greater kidney damage as indicated by elevated protein excretion (TG control vs. ETB def, 73.9±8.0 vs. 121.4±10.0 mg/day; n=5/group; p=0.01). In addition, ETB def rats fed HSD displayed cortical overexpression of 13 genes involved in energy metabolism, apoptosis, mitochondrial biogenesis, and regulation of the production of reactive oxygen species (selected examples of ETB def vs. TG control genetic expression (n=3/group): Ucp1, 19‐fold increase (p=0.03); Bnip3 (p=0.003) and Scl25a21 (p=0.004), 6‐fold; Mfn1,5‐fold (p=0.03); and Dnm1l, 4‐fold (p=0.004)). On the other hand, when the same comparisons were made in the kidney outer medullary region, we found that deficiency of the ETB receptor resulted in a 50% reduction in the expression of 29 genes (among them, genes involved in mitochondrial membrane polarization, transport and dynamics; p<0.05). These results demonstrate a differential effect of ETAoveractivation on mitochondrial homeostasis in the kidney cortical and outer medullary regions during high salt consumption, with overactivation of the ET‐1/ETA axis leading to exaggerated mitochondrial function in the cortex and downregulation of this function in the outer medulla.
Adipose tissue stiffness in the development of metabolic diseases
Authors: Tsai Yau‐Sheng , Huang Ann , Lin Yi‐Shiuan , Kao Ling‐Zhen , Tang Ming‐Jer , Tsai Pei‐Jane
Published: May 2022

Obesity, defined as an expansion of white adipose tissue, is also accompanied by interstitial fibrosis and overexpression of extracellular matrix (ECM) in adipose tissue in recent studies. We hypothesize that obesity induces adipose tissue ECM crosslinking and stiffening, leading to adipocyte dysfunction. First, we applied a collagen gel system to investigate the effect of ECM stiffness on adipogenesis and mature adipocyte functions. Our results showed that increased ECM stiffness impaired adipogenesis; and blunted the sensitivity to insulin and lipolytic cues, as well as secretion of adipokines, in differentiated adipocytes. In contrast, increased ECM stiffness exacerbated the sensitivity to inflammatory stimulation. Next, we found that ob/ob adipose tissue exhibited an increased signal in the polarized view of picrosirius red stained section, as well as increased crosslinks and aggregated collagen in second harmonics generation (SHG) imaging. These results suggest that ECM crosslinking was upregulated in the adipose tissue of obese mice. Ex vivo crosslinking inhibition in ob/ob adipose tissue explants increased insulin sensitivity, adiponectin expression and lipolytic activity. Finally, in vivo crosslinking inhibition reversed metabolic impairments and adipocyte functional signature genes in ob/ob mice. Taken together, our study underscores that adipose tissue crosslinking and stiffening may orchestrate metabolic disorders resulting from adipocyte dysfunction in obesity.
Functional correlates of lemur masticatory muscle fiber architecture visualized in 3D using DiceCT
Authors: Hartstone‐Rose Adam , Dickinson Edwin
Published: May 2022

The functional morphology of primate masticatory musculature has been the subject of many studies. However, with the exception of analyses of four taxa, these have always described this anatomy using traditional, destructive, gross anatomical dissections. In the current study, we use diffusible iodine‐based contrast‐enhanced computed tomography (DiceCT), in which specimens are stained with Lugol’s iodine that binds to the glycogen in muscle fibers to increase their radio‐opacity to the point of being able to discriminate between fascicles using x‐ray‐based imagery, to examine the masticatory muscle fiber architecture of nine strepsirrhine species across four genera of lemurid and Propithecus, Daubentonia and Otolemur. Not only do we report data on the traditional architectural variables of muscle volume, fascicle length (FL), physiological cross‐sectional area (PCSA), but also on variables unattainable except through three‐dimensional visualization: fascicular curvature and 3D orientation. The breadth of this sample more than triples the number of primates for which DiceCT has been applied to questions of fascicular architecture, allowing, for the first time, functional interpretation using this non‐destructive visualization technique. We have confirmed that this approach yields similar FL and PCSA measurements compared to those collected using destructive dissection. Further, this approach allows findings unavailable without this three‐dimensional visualization. Namely, fascicle angularity seems to relate to gouging – Daubentonia fascicles clearly align with its anteroposterior jaw motions. Further, fascicular compression seems relate to diet, with the most folivorous taxon in our sample, Propithecus, having 7% lower fascicular compression at near occlusion than that of Varecia, our most frugivorous taxon which has the highest arc:chord ratio (1.16) in the sample. While the scanning and subsequent “digital dissection” of the fascicles costs more time and money than does architectural data collection using traditional gross dissection, these expenses are coming down as scanners are becoming better and cheaper and digital tools (including improved selection algorithms) will allow faster fascicular analysis. Most importantly, these techniques not only yield data non‐destructively (allowing the study of specimens too precious to physically dissect), but they also preserve important three‐dimensional spatial relationships previously unavailable. This allows for the first analyses of true in situ fascicular orientation which is important for understanding naturalistic force vectors – a feature that may have important benefits for more accurate computer modeling of this complicated system – and fascicular compression – a feature with implications for understanding variable muscle activation during the chewing cycle – something previously only ever attainable with invasive live animal experimentation.
Protonation‐Dependent Sequencing of 5‐Formylcytidine in RNA
Authors: Link Courtney N. , Thalalla Gamage Supuni , Gallimore Diamond A. , Kopajtich Robert , Evans Christine N. , Nance Samantha R. , Fox Stephen D. , Andresson Thorkell , Chari Raj , Ivanic Joseph , Prokisch Holger , Meier Jordan L.
Published: May 2022

Chemical modification of cytidine in non‐coding RNAs plays a key role in regulating translation and disease. However, the distribution and dynamics of many of these modifications remains unknown due to a lack of sensitive site‐specific sequencing technologies. Here we report a protonation‐dependent sequencing reaction for detection of 5‐formylcytidine (5fC) and 5‐carboxycytidine (5caC) in RNA. First, we evaluate how protonation combined with electron withdrawing substituents alters the molecular orbital energies and reduction of modified cytidine nucleosides, highlighting 5fC and 5caC as reactive species. Next, we apply this reaction to detect these modifications in synthetic oligonucleotides as well as endogenous human tRNA. Finally, we demonstrate the utility of our method to characterize a patient‐derived model of 5fC‐deficiency, where it enables facile monitoring of both pathogenic loss and exogenous rescue of NSUN3‐dependent 5fC within the wobble base of human mitochondrial tRNAMet. These studies showcase the ability of protonation to enhance the reactivity and sensitive detection of 5fC in RNA, and provide a molecular foundation for applying optimized sequencing reactions to better understand the role of oxidized RNA cytidine nucleobases in disease.
Metabolic Profile in Neonatal Pig Hearts
Authors: Li Pengsheng , Tang Ling , Zhu Wuqiang
Published: May 2022

Background

Cardiomyocytes in postnatal hearts undergo hypertrophy to adapt to increased blood pressure and volume. The process of cardiomyocyte maturation is associated with a cessation from cell cycle and a series of metabolic switches. Prior research has shown metabolic switches in developing mouse hearts. However, metabolic changes in postnatal pig hearts have not been reported.

Methods

We evaluated the metabolic profile in pig hearts at postnatal day 1 (P1), day 3 (P3), day 7 (P7), and day 28 (P28) (n=6 each, both males and females; Yorkshire‐Landrace Cross background) using a targeted liquid chromatography tandem mass spectrometry assay. Expression of lactate dehydrogenase isoforms was evaluated by western blotting.

Results

There is a clear separation of the detected metabolites in P1 versus P28 hearts. Active anabolisms of nucleotide and proteins were observed in P1 hearts when cardiomyocytes retain high cell cycle activity. Abundance of the intermediate metabolites in the pathways of glycolysis was decreased in P28 comparing to P1 hearts, which implicates an overall reduced level of glycolysis. The decreased abundance of intermediates in TCA cycle and increased abundance of lactic acid in P28 hearts suggested that glycolysis rather than glucose oxidation is the main form of glucose metabolism in mature pig hearts. Reduced abundance of GLCN6P was observed in P28 hearts compared to P1 hearts. Increased abundance of hydroxyproline was observed in P1, P3, and P7 hearts compared to P28 heart.

Conclusion

Postnatal cardiomyocyte maturation is associated with metabolic switch from glucose to fatty acids, active posttranslational protein modification and ECM remodeling.
An Application of Mycobacteriophage Genome Engineering using Bacteriophage Recombineering with Electroporated DNA (BRED) and CRISPR Cas‐9 Systems
Authors: Dionne Ethan N. , Cornely Kathleen
Published: May 2022

The increasing occurrence of antibiotic‐resistant pathogens remains a global threat to public health, spurring research to develop alternative treatments for infectious bacteria. Phage therapy, the targeted use of bacteria‐specific viral particles called bacteriophages, has emerged as a promising antibiotic alternative. Our lab studies mycobacteriophages – phages specific to bacteria of the genus Mycobacterium,which includes infamous, antibiotic‐resistant pathogens such as M. tuberculosisand M. abscessus.For the use of many mycobacteriophages in phage therapy treatments, gene deletions are often necessary to ensure host lethality. Previous methods for mutant generation used a technique called bacteriophage recombination with electroporated DNA (BRED), which used electrocompetent host cells equipped with recombination machinery, genomic phage DNA, and a synthetic DNA g‐block to generate mutants. This process proved inefficient with recalcitrant recovery of some deletions, requiring extensive screening of plaques. To improve the process of genetic modifications with mycobacteriophages, greater precision and improved screening has recently been achieved through the combined use of BRED with a mycobacteria‐efficient Streptococcus thermophilus CRISPR Cas‐9 gene knockdown system for selection against the parent phage (CRISPY‐BRED). Here we present an application of the CRISPY‐BRED technique for the deletion of the immunity repressor gene in the mycobacteriophage known as Mufasa. The Cas‐9 system involves a plasmid containing the modified, nuclease‐dead Cas9 gene, a sgRNA fused to an anhydrotetracycline inducible promoter, a kanamycin resistance gene, an E. colireplication origin, and an attPsite for chromosomal integration in mycobacteria. The identification of a Protospacer Adjacent Motif (PAM) within the mycobacteriophage immunity repressor gene allowed for the design of a 20 bp sgRNA targeting sequence from the phage DNA. With host cell transformation of this CRISPR Cas9 plasmid programmed to target the parent phage immunity repressor and inhibit overall parent phage gene expression, straightforward mutant recovery should be achievable.
Reporting COVID‐19 Cases within Kleberg County
Authors: Lopez Laura , Velez‐Hernandez Maria
Published: May 2022

A global pandemic due to the coronavirus‐2019(COVID‐19) was declared in March 11 of 2020. The RNA COVID‐19 virus is part of the coronaviridae family and causes severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The progression of the disease can lead to respiratory issues that necessitate mechanical assistance, such as ventilation and intensive care. Daily reports of COVID‐19 cases informed citizens of the severity of the pandemic in their area and in many cases, guided government responses like masks mandates and lockdowns. This study investigates the reporting of COVID‐19 cases in Kleberg County, Texas, USA. Data reported in social media by Kleberg County from July 2020 to April 2021 is compared to COVID‐19 cases reported by the The New York Times. This study also includes a further look at case distribution between males and females in Kleberg County. Results indicate that The New York Times reported 793 more cases than Kleberg County during this time and there were 36 more male than female cases. Transparent and accurate data reporting is crucial for public health efforts. The disparity in COVID cases highlights the need for better reporting of accurate data and reflects the inaccurate and sometimes inadequate response to the pandemic in Kleberg County.
Modeling Structural Adaptation and Pruning of Blood Vessels Connected in Parallel: Implications for Control of Capillary Density
Authors: Nguyen Vivian T. , Secomb Timothy W.
Published: May 2022

Blood vessels are capable of structural adaptation in response to changes in physiological conditions including blood flow rates and oxygen levels. A theoretical model is developed to simulate the changes in diameter of two neighboring microvessels connected in parallel. The model is used to examine the conditions leading to stability of this configuration or to pruning of one of the vessels. When two adjacent vessels supply blood to the same region of tissue, structural adaptation is governed by two competing mechanisms: the elimination of a redundant vessel to minimize the energy cost of sustaining blood flow, and the stabilization of both vessels to distribute oxygen more broadly. Therefore, the behavior of the system depends on both the strength of the metabolic oxygen demand from the tissue and the distance between the vessels. In the simulation, the diameters of two vessels connected in parallel vary based on the mechanical shear and pressure forces acting on the endothelial wall, an oxygen‐dependent metabolic stimulus, and a constant tendency for vessels to shrink. The distribution of oxygen throughout the tissue is calculated using a Green’s function method. An initial condition is assumed in which the vessels have near equal diameters. The strength of the metabolic response needed to stabilize both vessels with equal diameters is determined as a function of the distance between the vessels. In cases where equal diameters are unstable, the condition for pruning of one of the vessels, defined as a diameter below 3 μm, is found. For a given spacing between the vessels, two critical values are found for the strength of the metabolic signal. Above the higher critical value, the two vessels are stable with equal diameters. Between the critical values, the two vessels approach a state with unequal diameters. The ability of vessels to stabilize at unequal vessel diameters may contribute to the heterogeneity of physiological vasculatures. Below the lower critical value, one of the vessels is pruned. Under the assumed conditions, both critical values decrease with increasing spacing up to about 70 μm, and are independent of spacing for larger distances. For a given strength of the metabolic signal, smaller spacing favors pruning of one of the vessels because their proximity allows one vessel to meet the metabolic demand of the tissue. The pruning of closely spaced vessels may contribute to the control of capillary density in tissues such as skeletal muscle. Further work is needed to determine whether similar behavior occurs in arrays of multiple capillaries as in muscle vasculature.
Control of glycosyltransferase activity by nucleotide‐binding active site residues in SECRET AGENT
Authors: Philbrick Kaylee , Nelson Dana , Dallmier Kate , Chalfant Kaitlyn , Olson Brian , Joiner Cassandra
Published: May 2022

Human O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) is the only glycosyltransferase that operates in the nucleus and cytoplasm, targeting over 1,000 unique substrates. OGT is conserved across all eukaryotic species, and misregulation of protein O‐GlcNAcylation has been implicated in cancers and neurodegenerative diseases. Secret Agent (SEC) is a structural homolog of OGT in Arabidopsis thaliana and like OGT, it consists of two comparably‐sized domains: an N‐terminal tetratricopeptide repeat (TPR) domain that mediates protein‐protein interactions and a C‐terminal catalytic domain that mediates O‐GlcNAc transfers. Both SEC and OGT transfer GlcNAc from UDP‐GlcNAc to serine and threonine residues on their substrates, but the exact mechanisms of SEC nucleotide‐sugar selection and catalysis are unknown. In the catalytic domain, UDP‐GlcNAc binds directly to OGT and makes several key contacts to active site residues before protein substrates bind over the nucleotide‐sugar, making nucleotide‐sugar binding essential for catalysis. To determine whether OGT’s mechanisms of nucleotide‐sugar selection and catalysis are conserved in the SEC homolog, we investigated residues in the SEC active site corresponding to known essential residues in OGT that make key interactions with the UDP nucleotide portion of UDP‐GlcNAc. Mutant SEC protein was purified from E. coli and analyzed for catalytic activity. Active site mutations known to inhibit OGT activity were also found to reduce activity in SEC, suggesting a conserved mechanism for nucleotide‐sugar selection.