bluuebunny/crossref_metadata_2025 huggingface dataset:
TRPM4 Ion Channels in Glycemic Control during Pregnancy
Authors: Wuyts Caroline , Philippaert Koenraad , Pinto Silvia , Simoens Caroline , Vennekens Rudi
Published: May 2022

During pregnancy, the glucose homeostasis of the mother adapts to accommodate the needs of a growing fetus. Decreased insulin sensitivity and increased insulin secretion from the pancreatic beta‐cell contribute to this adaptation. Electrophysiological changes in the beta‐cell during pregnancy, that could explain the increased insulin secretion, have not yet been identified. The non‐selective Transient Receptor Potential Melastatin 4 (TRPM4) cation channel, expressed in beta‐cells, may depolarize the beta‐cell membrane and promote insulin release. In other organs, TRPM4 ion channel expression fluctuates depending on the relative concentration of estrogen and progesterone, hormones important during pregnancy. These findings suggest a role of TRPM4 in glycemic control, which can be influenced by female reproductive hormones during pregnancy.

Using in vivo continuous glucose monitoring throughout the full pregnancy of wildtype (WT) and TRPM4 knockout (KO) mice allows to analyze glycemia during oral glucose tolerance tests (OGTTs) and spontaneous feeding behavior, and to determine glycemic variability. TRPM4 KOmice are hyperglycemic compared to WT mice, mainly before and during the first half of pregnancy. The mean glucose concentration was increased in TRPM4 KO mice in the beginning of pregnancy. In both WT mice and TRPM4 KO mice, the overall glycemia gradually increased during this period, maintaining the difference in glycemia between the two genotypes. Later in pregnancy, the glycemia of both genotypes started gradually decreasing. However, glycemia of TRPM4 KO mice decreased faster, reducing the difference in glycemia between the two genotypes. This indicates a different adaptation to pregnancy. The maximum glucose levels reached during the OGTTs were larger for the TRPM4 KO mice compared to the wildtype mice, which is in line with the general observed hyperglycemia of TRPM4 KOmice. Furthermore, we observed that fasting glucose concentrations, obtained right before the OGTTs, were significantly increased for the TRPM4 KO mice compared to WT mice. Glycemic variability provides information regarding sudden changes and fluctuations in the blood glucose concentration. In general, there were no substantial differences in terms of glycemic variability between both genotypes. To conclude, we could show that TRPM4 plays an active role in glycemic control, before and during pregnancy.
Effects of Dual‐Species Biofilm Formation on Plastic Degradation by Ideonella sakaiensis
Authors: Williams Tawnie B. , Russell Andrew R. , Mitchell Jon C.
Published: May 2022

As of 2015, nearly 80% of the world’s virgin plastic ended up in a landfill or the natural environment, a majority accredited to the packaging industry. Due to the composition of the durable plastic, polyethylene terephthalate (PET), the primary polymer found in single‐use packaging, is not naturally biodegradable and thus accumulates. Recently, several microbes were discovered that could degrade the plastic compounds within their gut with no signs of toxicity. Ideonella sakaiensisis a novel bacterium capable of depolymerizing PET using a hydrolase termed PETase. An additional enzyme, MHETase, aids in converting dimers for the complete recovery of monomers within I. sakaiensis. Ultimately, efficiency in degrading PET of varying crystallinity occurs with a preference of low crystallinity PET, in opposition to other species currently known. During degradation, I. sakaiensisforms a thin biofilm over the plastic. It is known that the cells contain special appendages to adhere to the plastic surface, however, little research has been conducted on how the biofilm formation affects the degradation process. Multi‐species biofilms have been shown to create complex communities for enhanced PET degradation in marine plastic debris using other bacterial strains.

This research aims to study the biofilm formation as well as its effects on its degradation properties of I. sakaiensisas an isolate, as well as a dual species biofilm with known biofilm forming bacteria: Stenotrophomonas maltophilia, Staphylococcus aureus, and Pseudomonas aeruginosa. The first half of the project characterized the effects of introducing biofilm forming bacteria on I. sakaiensis’ biofilm growth. The second half of the project analyzes the biofilm growth along with a degradation assay to determine the effects of these bacterial strains on the degradation process of I. sakaiensis. These effects were then evaluated to determine the role of biofilm formation in the degradation process. This study provides a foundation that can be used by the biotech, environmental, and molecular communities to lead to a commercialization of plastic degradation products, ultimately creating a circular and stable economy within the consumer world.
Dividing Cells are Most Susceptible to Targeted Osmotic Lysis Cancer Therapy
Authors: Edenfield Samantha , Lemoine Spencer G. , Gould Harry J. , Paul Dennis
Published: May 2022

Voltage‐gated sodium channels (VGSCs) are upregulated in aggressive carcinomas, making these channels a target for novel clinical therapies. Targeted Osmotic Lysis (TOL) is a novel cancer therapy that combines pharmacologically blocking Na+,K+, ATPase (sodium pumps) while stimulating VGSCs with a pulsed electric field. The consequent increase in intracellular Na+ causes an osmotic lysis of the cells that overexpress VGSCs. Noncancerous cells, with fewer VGSCs do not lyse. We hypothesized that variability in efficacy of TOL is due to VGSC expression that varies across the cell cycle. We assessed cell surface expression of VGSCs and Na+,K+‐ATPase during phases of the cell cycle in which there are single copies of DNA compared with phases in which DNA has doubled. For this, DNA was labeled with either propidium iodide (PI) or DAPI and VGSCs were labeled with a pan‐specific VGSC antibody while Na+,K+‐ATPases were labelled with a pan‐specific Na+,K+‐ATPase antibody in eight immortalized cancer and non‐cancer cell lines. With flow cytometry we showed that VGSC expression doubled during phases of cell division (S‐M phases) compared to G0‐G1 phases, whereas Na+,K+‐ATPase expression increased by 1.5‐fold. To investigate whether cells in S‐M phases are more susceptible to TOL due to the increase in VGSCs, cells were treated and immediately fixed and stained with PI for cell cycle analysis. There was a 30% decrease in the percentage of total cells that were treated with TOL compared to cells that did not receive TOL treatment. This finding is evidence that dividing cells are more susceptible to TOL than cells in G0‐G1 phases. It is thus likely that combined therapies will likely lead to better patient outcomes.
Characterization of Novel, Agmatine‐Based NMDA Receptor Antagonists in Multiple Pain States
Authors: Peterson Cristina , Kitto Kelley F. , Wilcox George L. , Fairbanks Carolyn
Published: May 2022

The worsening opioid epidemic has highlighted the need for the development of new, safe, and effective analgesic therapeutics. Opioid therapy currently is associated with the risk of conversion to addiction, diversion from patients for whom use is intended, and the development of analgesic tolerance, or the loss of efficacy with continued treatment. To this end, we have developed a line of non‐opioid, agmatine‐based compounds and assessed them for their efficacy in reversing behavioral expressions of pain in animal models, as well as evaluated their safety following chronic exposure. We have previously shown that agmatine, decarboxylated L‐arginine, is an N‐methyl‐D‐aspartate (NMDA) receptor antagonist that preferentially antagonizes receptors that express NR2B subunits. This preferential antagonism is desirable for NMDA‐based therapeutics as it can lead to a widening of the therapeutic window and avoidance of the side effects commonly associated with NMDA antagonism including motor ataxia and psychoactive effects. However, agmatine has shown limited penetration through the blood brain barrier (BBB) and a short systemic half‐life, limiting its clinical utility. We have designed strategically‐substituted agmatine (SSA) compounds with the goal of improving its penetration through the BBB by increasing the lipophilicity of agmatine, potentially improving distribution across the BBB and increasing its half‐life following systemic delivery. To this end, we have evaluated this series of SSAs for safety and efficacy in multiple animal models of pain.

Mice (21‐30 g, M/F) were assessed for their baseline mechanical sensitivity, and then one of several models of pain was induced: inflammatory pain (Complete Freund’s Adjuvant, (CFA) injected into a hindpaw), neuropathic pain (spared nerve injury surgery), or post‐surgical pain (hindpaw muscle incision). Mechanical sensitivity was again assessed, then an SSA compound, agmatine (the parent compound), or vehicle control was administered, and mechanical sensitivity was recorded for up to three hours following administration. The SSA compounds effectively reversed pain behaviors in mice following administration in the various pain models. Additionally, side effects characteristic of NMDA receptor antagonists were assessed and not found at the range of doses that produced analgesia, indicating a wide therapeutic window.

These data indicate that the strategically‐substituted agmatines are anti‐hyperalgesic compounds with a wide therapeutic window, avoiding the motor impairment typical of drugs of this class.
Growth and differentiation of human induced pluripotent stem cell (hiPSC) derived kidney organoids using fully synthetic peptide hydrogels
Authors: Treacy Niall J. , Clerkin Shane , Davis Jessica L. , Kennedy Ciarán , Wychowaniec Jacek K. , Brougham Dermot F. , Crean John
Published: May 2022

Human induced pluripotent stem cell (hiPSC)‐derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine. However, there remains a necessity to refine the biophysical and biochemical parameters that govern kidney organoid formation. Differentiation within fully‐controllable and physiologically relevant 3D growth environments will be critical to improving organoid reproducibility and maturation. Here, we matured hiPSC‐derived kidney organoids within fully synthetic self‐assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G′). The resulting organoids contained complex structures comparable to those differentiated within the animal‐derived matrix, Matrigel. Single‐cell RNA sequencing (scRNA‐seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air‐liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Organoid compositional analysis revealed a larger proportion of nephron cell types within Transwell‐derived organoids, while SAPH derived organoids were enriched for stromal‐associated cell populations. Notably, differentiation within a higher G’ SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of offtarget cell types, which are a known limitation of current kidney organoid protocols. This work demonstrates the utility of synthetic peptide‐based hydrogels with a defined stiffness, as a minimally complex microenvironment for the selected differentiation of kidney organoids.
Transcriptome‐Metabolome Association Studies in Mouse Lungs Reveal Differences Between Sex and Strain in the Glutathione Antioxidant Pathway
Authors: Rogers Lynette K. , Fernandes Jolyn , Dunnigan‐Russell Katelyn K. , Zhong Hua , Lin Vivan , Silverberg Mary , Moore Stephanie , Li Qian , Li Rui , Vitiello Peter F. , Tran ViLinh , Jones Dean P. , Tipple Trent E.
Published: May 2022

Sex and strain significantly influence pathophysiological responses to environmental exposures in mice. Underlying sex and strain differences have not been clearly investigated but are essential to understanding unique molecular and pathological features of experimental pulmonary disease models. We used an integrated transcriptome and metabolome approach to test for sex and strain‐based differences in functional pathway and network responses in lungs of C3H/HeN and C57Bl6 mice. Results from multivariate analysis at P< 0.05 and FDR at 0.05 revealed differentially expressed metabolites (m) and transcripts (t) between males and females (660 m, 484 t), C3H/Hen and C57Bl6 strains (921 m, 418 t) and as a result of interaction between sex and strain (374 m and 383 t). Metabolic pathway analysis using mummichog showed drug metabolism was the most common pathway affected by sex, strain and interaction. Oxidative stress related pathways such seleno amino acid metabolism reflected strain‐based influence while cysteine/glutathione pathways were influenced by sex and the combined interaction of sex and strain. Gene set enrichment analysis revealed inflammatory response as the underlying differential pathway in sex, strain and interaction‐based transcriptomic differences and several antioxidant genes were identified including glutamatecysteine ligase C, catalase, and peroxiredoxin 1. Overall, these results suggest that underlying metabolic and transcriptomic differences in antioxidant regulation and oxidation of key molecules may be responsible for the differences observed in sex and strain‐based pulmonary susceptibilities.
The Oncogene MYC Regulates the Branched Chain Amino Acid Metabolism and mTOR Signaling in Diffuse Large B Cell Lymphoma
Authors: Follett Aleyna , Figueroa Lucas , Boyer Michael , Ananieva Elitsa
Published: May 2022

Diffuse large B‐cell Lymphoma (DLBCL) is the most common type of aggressive non‐Hodgkin lymphoma (NHL) worldwide. While therapeutic strategies to combat DLBCL have advanced, these malignancies continue to be challenging to treat due to their heterogeneity and frequently recurring mutations. Aberrations in the oncogene MYC are commonly found in high grade DLBCL and are associated with poor responses to standard therapy.

In search of downstream targets of MYC in DLBCL, we found MYC‐binding sites in the promoter regions of BCAT1, BCAT2, and BCKDHA. These genes encode enzymes in the metabolism of the branched chain amino acids (BCAAs), and they have been previously linked to cancer progression. BCAT1 and 2 encode the cytosolic (BCATc) and mitochondrial (BCATm) branched‐chain aminotransferases, while BCKDHA encodes the E1α subunit of the branched‐chain ketoacid dehydrogenase complex. We hypothesized that MYC activates these genes in DLBCL tissues leading to upregulation of BCAA metabolism and lymphoma growth.

Human tissues collected from DLBCL patients (n=89) or controls (n=33) were used to analyze the gene expression of MYC, BCAT1, BCAT2 and BCKDHA followed by correlative analysis of the expression of these genes and the survival of DLBCL patients. Next, human DLBCL and B‐lymphoblast (control) cells were treated with the MYC inhibitor 10058‐F4 (0, 25, 50, 100 µM), the BCAA antagonist N‐acetyl leucine amide (NALA, 0, 10, 20, 40 mM) and the BCATc inhibitor gabapentin (0, 5, 10, 20 mM). 24 hours post treatment, the cell viability and cell proliferation, as well as the protein expression of BCATc, BCATm, and E1α, and the total and phosphorylation states of the ribosomal protein S6 (a downstream target of the mammalian target of rapamycin, mTOR) were determined.

Results showed that the gene expression of BCAT1, BCAT2and BCKDHA were significantly elevated in tumor tissues from DLBCL patients compared to controls, and there was a positive correlation between their expression and the upregulation of MYC in DLBCL tissues. The high expression of MYC and BCAT2, but not that of BCAT1 and BCKDHA, correlated with poor overall cancer survival. Inhibiting the MYC function by 10058‐F4, the BCAA uptake by NALA, and the BCATc activity by gabapentin led to concentration dependent reductions in the cell viability and proliferation of the DLBCL cells. The protein expression of BCATm and E1α were reduced in the presence of 10058‐F4, which correlated with downregulation of mTOR signaling as measured by the deceased phosphorylation state of S6 in treated DLBCL cells. These results support the hypothesis that MYC is an upstream regulator of genes associated with BCAA metabolism. The results also demonstrate that DLBCL cells need MYC and BCAAs to upregulate mTOR signaling and to survive and may represent a new molecular mechanism via which MYC exerts its effects in DLBCL lymphoma.
2D Geometric Morphometric Shape Analysis of Casuarius casuarius(Aves: Paleognathae) Cranial Casques
Authors: Green Todd L. , Kay David I. , Gignac Paul M.
Published: May 2022

Many vertebrates (e.g., chameleons, bovids, hornbills) possess cranial elaborations; however, studies investigating the morphological variation of these structures are rare. For example, the conspicuous cranial casques of modern cassowaries have long been hypothesized to perform a number of functions (e.g., physical ramming, vocalization, thermoregulation, visual display) despite an absence of quantitative methodologies to determine whether ornament shape variation within the group aligns with proposed biological roles. In order to fill this data gap, we compared casque shape between sexes and native geographic regions in a large sample of adult southern cassowaries (Casuarius casuarius; n = 103). We quantified casque shape in lateral and rostral views using elliptical Fourier analysis, ordinated the shape data, and used multivariate analysis of variance to address specific comparisons. We hypothesized that casques would be sexually dimorphic and regionally specific, consistent with proposed display functions. However, we found no significant differences in casque shape between female and male C. casuariusand few significant shape differences between individuals from specific geographical areas. Much of the intraspecific casque shape variation, particularly from the rostral aspect, is due to asymmetries across the midline. In our sample, casques were 77.5% rightward deviating, 20.7% leftward deviating, and 1.8% non‐deviating. Although it is unclear if this directional casque asymmetry provides a functional advantage, future studies may elucidate this feature. Altogether, two‐dimensional shape analysis does not support a role for the casque as a de factodiscriminator between sexes nor regional sub‐populations in C. casuarius. These findings stand in contrast to those of other ornamented birds (e.g., Numida meleagris) and potentially point towards alternative biological functions.
P‐gp and Bcrp Exert Differential Impacts on Maternal and Fetal Exposure to Tetrahydrocannabinol and Its Major Metabolites in Pregnant Mice
Authors: Chen Xin , Unadkat Jashvant D. , Mao Qingcheng
Published: May 2022

In the United States, cannabis consumption is increasing. (‐)‐Δ9‐tetrahydrocannabinol (THC) is the primary pharmacological active constituent of cannabis, while 11‐hydroxy‐THC (11‐OH‐THC) and 11‐nor‐9‐carboxy‐THC (THC‐COOH) are active and nonactive circulating metabolites of THC in humans, respectively. P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are ATP‐binding cassette efflux transporters important for drug disposition, including brain penetration and fetal exposure. Our previous in vitro transport study showed that only THC‐COOH is a weak substrate of human BCRP, while previous animal studies reported that THC and its metabolites could be substrates of the P‐gp and BCRP orthologs in mice and macaques. To further investigate the roles of P‐gp and BCRP in the maternal‐fetal disposition of THC and its metabolites, we administrated 3 mg/kg THC (retro‐orbitally) to FVB wild‐type (WT), P‐gp‐/‐, and Bcrp‐/‐ pregnant mice on gestational day 18, and determined maternal, placental, and fetal concentrations of THC and its metabolites by LC‐MS/MS. The AUCs of THC and its major metabolites were estimated using a pooled data bootstrap approach. Differences in tissue/maternal plasma AUC ratios between WT and P‐gp‐/‐ or Bcrp‐/‐ were compared via unpaired parametric t‐test, and differences with p values of < 0.05 were considered statistically significant. In Bcrp‐/‐ pregnant mice, the placenta/maternal plasma AUC ratio for THC was 188% (p < 0.05) of respective AUC ratio in WT pregnant mice. In P‐gp‐/‐ pregnant mice, the maternal brain/maternal plasma AUC ratio for THC, maternal brain/maternal plasma and fetus/maternal plasma AUC ratios for 11‐OH‐THC, and fetus/maternal plasma AUC ratio for THC‐COOH were 29%, 65%, 49%, and 54% (p < 0.05) of respective AUC ratio in WT pregnant mice. There were no significant differences in fetal exposure of THC in either P‐gp‐/‐ or Bcrp‐/‐ mice, and in fetal exposure of 11‐OH‐THC and THC‐COOH in Bcrp‐/‐ mice, compared to WT mice. In conclusion, this study revealed that P‐gp and Bcrp exerted differential impacts on maternal brain, placental, and fetal exposure to cannabinoids in pregnant mice. P‐gp and Bcrp do not seem to play a role in limiting fetal exposure to THC and its major metabolites; but could differentially alter maternal brain exposure to the cannabinoids.
Obesity Exacerbates Lipid Nephrotoxicity and Renal Injury in a Model of Ovarian Hormone Deficiency
Authors: Walsh Patrick T. , Johnson Scott M. , Kim Woo Suk , Babich Victor , Barnes Maria J. , Di Sole Francesca
Published: May 2022

Obesity rates are continually increasing both in the USA and around the world. As rates of obesity continue to grow, public health concerns remain a top priority in order to curtail mortality and morbidity. An increase in visceral adipose tissue is correlated with dysfunction in multiple organs, including the kidneys. Increases in basal metabolic index, or BMI, has been linked to increases in triglyceride accumulation in the kidneys. Obesity accounts for ~25% of kidney diseases observed in women; however, its effects on kidney function have not been defined in post‐menopausal women. In an ovarian hormone deficient mouse model (ovariectomized animals), we measured a significant weight gain compared with sham‐operated control, associated with perirenal adipose tissue accumulation, and an increase in 1. glomerular filtration rate (GFR) used to determine renal function, 2. protein level of the NGAL, an early biomarker of kidney injury and 3. intrarenal lipid accumulation found in the renal tubules. In this study, we aim to determine whether obesity affects the levels of renal injury and lipid accumulation measured in a model of ovarian hormone deficiency. We studied four groups of mice: the first and second groups underwent ovariectomy (OVX); group one was fed a 60% high‐fat (HF) diet, and group two was on a 10% low‐fat (LH) diet. The third and fourth groups (controls), underwent surgery without ovariectomy (SHAM) and were fed a HF or LF diet. All animals had ad libitum access to their respective diet for fourteen weeks (i.e., ten weeks prior to surgery and four weeks after). Significant increase in body weight was measured in mice on HF compared to LF diet. The size of triglyceride droplets in the renal tubules was significantly higher (20‐25%) in OVX compared to SHAM‐operated control mice on both HF and LF diets. The Neutrophil Gelatinase Associated Lipocalin (NGAL) protein levels were measured as a biomarker of kidney injury. Levels of NGAL protein expression increased in OVX mice when compared to SHAM control in the renal tubules of mice on both HF (211.3 ± 3.7%, p<0.0001) and LF diet (111.33 ± 1.7%, p=0.02). In the glomeruli, NGAL protein expression levels increased in OVX mice compared to SHAM control in HF (188.8 ± 9.9%, p<0.0001) and LF diet fed groups (113.1 ± 4.9%, p=0.64). Furthermore, mice on HF diet had significant higher NGAL levels in OVX and sham‐control conditions than mice in the same conditions on LF diet. These findings indicate that weight gain exacerbates renal injury induced by OVX, and that estrogen may be protective against excessive accumulation of triglycerides in kidney structures. Our study supports the understanding of the mechanisms implicated in the development of kidney diseases in obese post‐menopausal women.
Investigating β‐Lapachone Mediated Metabolic Disruption Using Stable Isotope Tracers
Authors: Chang Mario C. , Mahar Rohit , Merritt Matthew E.
Published: May 2022

β‐lapachone has been found to be a highly potent anti‐cancer agent in phase 1 clinical trials, but has dose limiting toxicity in red blood cells [Gerber et. al Br J Cancer. 2018 Oct;119(8):928‐936]. Identifying a safe and quantitative method of monitoring treatment efficacy will be highly beneficial to establishing an adaptive dosing platform. Adaptative dosing can allow personalized treatments that significantly reduce off target effects while maintaining an effective therapeutic regimen. In this study we present the use of a safe and noninvasive tracer strategy utilizing both [2H7]glucose and [U‐13C]glucose tracers to assess the metabolic effects caused by β‐lapachone treatment in vitro. Treating cancers with β‐lapachone bioactivates NAD(P)H: quinone oxidoreductase 1 (NQO1) to produce a highly unstable hydroquinone intermediate that reestablishes itself through an aggressive futile cycle, generating reactive oxygen species (ROS) in the form of hydrogen peroxide. Accelerated accumulation of ROS causes large amounts of DNA damage, triggers poly‐ADP‐ribose polymerase‐I hyperactivation, induces global depletion of NAD+ and ATP, and hinders overall glycolytic flux [Moore et. al Cell Death Dis. 2015 Jan; 6(1): e159]. Cancer cells that overexpress NQO1 subsequently die through NAD+‐keresis. Measuring changes in glycolysis and downstream metabolism caused by the bioactivation of NQO1 would establish a platform for assessing the efficacy of treatment, potentially allowing the reduction of chemotherapeutic dosage thereby diminishing off‐target toxicities. Changes in central carbon metabolism caused by NQO1 bioactivation were detected and assessed in various cancer cells using both tracer strategies. Deuterated lactate and HDO (deuterated water) were easily quantified, and the production of these metabolic products were linearly correlated with [2H7]glucose consumption. Mass isotopologue distribution analysis by gas chromatography‐mass spectrometry demonstrated downregulated energy metabolism with a reduction in 2H and 13C labeling in NQO1+ cancer cells treated with β‐lapachone. These methods are highly beneficial for measuring rates of glycolytic metabolism, identifying potential therapeutic synergies, the efficacy of chemotherapeutic agents that hamper central carbon metabolism, and can be potentially translated to in vivo models.
Pentadecapeptide BPC 157 Counteracts Hemodynamic and ECG Disturbances Induced by Sotalol in Rats
Authors: Premuzic Mestrovic Ivica , Smoday Ivan Maria , Mestrovic Tomislav , Zizek Helena , Skrtic Anita , Skorak Ivan , Haluzan Damir , Figl Josip , Brizic Ivan , Staresinic Mario , Pavic Predrag , Seiwerth Sven , Sikiric Predrag
Published: May 2022

Cytoprotective and organoprotective pentadecapeptide BPC 157 demonstrated particular beneficial cardiovascular effects (Curr Pharm Des. 2014;20(7):1121‐1125). Antiarrhythmic effects counteracted digoxin toxicity (Regul Pept. 2009;156(1‐3):83‐89), major blood vessel occlusions (Biomedicines 2021, 9,1029; 2021, 9,609; 2021, 9, 792) and lidocaine toxicity (Emerg Med Int. 2020;2020:6805354). Now, we reveal, by BPC 157, mitigation of hemodynamic and ECG disturbances of sotalol, a class III antiarrhythmic with non‐selective beta‐blocking activity.

Albino Wistar rats received sotalol (80mg/kg, intragastrically), and at 5 min thereafter were treated with saline or BPC 157 (10ng/kg, intragastrically. Seven standard and one precordial ECG leads were recorded (baseline; after application of sotalol; and 10, 30, 90, 120 and 150 minutes after application of sotalol). Heart rate, QRS complex, PR and QT intervals were analyzed. Corrected QT intervals (QTc) were calculated according to Fridericia formula.

Baseline ECG readings included average heart rate of 275.5 ± 25 bpm, average PR 54.8 ± 3.4 ms, average QRS complex 25.0 ± 4.23 ms and average QTc 166 ± 9.7 ms. Sotalol caused bradycardia (245 ± 23.5 bpm), PR prolongation (57.3 ± 2.7 ms), QRS widening (26.3 ± 3.2 ms), and QTc prolongation (173 ± 9.1 ms), measured 10 minutes after application. In BPC 157 treated animals (5 minutes after BPC‐157 application, 10 minutes after sotalol application), these effects were attenuated (heart rate 306 ± 31.7 bpm, average PR 52.3 ± 1.6 ms, average QRS 23.7 ± 5.4 ms and average QTc 159 ± 21.2 ms). This attenuation was lost in later measurements, for QRS ‐ 25 minutes after BPC 157 application, for frequency after 55 minutes, for PR after 115 minutes and for QTc after 145 minutes. In animals treated with sotalol, an increase in superior sagittal sinus pressure (10 ± 5 mmHg), as well as an increase in portal vein (17 ± 4 mmHg) and inferior caval vein pressures (12 ± 6 mmHg) were noted, along with the decrease in abdominal aortic pressure (53 ± 10 mmHg). These effects were attenuated in BPC treated animals (‐1 ± 1 mmHg for superior sagittal sinus, 7 ± 1 mmHg in portal vein, 5 ± 1 mmHg in inferior caval vein, and 81 ± 7 mmHg in abdominal aorta).

In conclusion, BPC 157 attenuated deleterious hemodynamic effects of sotalol while the effects on the ECG parameters were also noted, although not long lasting, supporting the view of BPC‐157 as a rapid‐acting protective agent.
Central Command Related Changes in Anterior Cerebral Artery Blood Velocity and Prefrontal Oxygenation at the Onset of Moderate Voluntary Hand‐Grip Exercise
Authors: Adesanya E.A. Omoluyi , Matsukawa Kanji , Young Benjamin E. , Trotter Claire E. , Davis Scott L. , Jarrad Caitlin P. , Huang Mu , Smith Scott , Moralez Gilbert
Published: May 2022

Recently, it has been identified that volitionally initiated finger tapping, which is a “very light motor effort” physical activity, elicits greater increases in mean anterior cerebral artery blood velocity (ACAmean), ACA vascular conductance index (ACAvci) and prefrontal oxygenation (Oxy‐Hb) responses compared to a cued start. These findings suggest that differences in ACAmean and prefrontal Oxy‐Hb responses between the exercise initiation modes may be related to central command. However, it remains unknown if similar responses are observed at the onset of voluntary exercise of a “moderate motor effort”. Therefore, the purpose of this study was to determine ACAmean, ACAvci and prefrontal Oxy‐Hb responses during dynamic hand grip exercise at 40% of maximal voluntary contraction (MVC) during two exercise initiation modes (voluntary or cued start). ACAmean, ACAvci, and prefrontal Oxy‐Hb were measured in seven participants (4 females and 3 males; aged 26 ± 3 years) at baseline (‐30 sec to ‐20 sec prior to initiation of exercise), pre‐exercise (‐20 sec to 0 sec) and the onset of exercise (0 sec to 15 sec) of each exercise initiation condition. Participants perceived exertion Borg Scale rating for the 40%‐MVC was 11.5 (median). A two‐way repeated measures ANOVA was conducted for the variables of interests. A significant factor interactions was identified for ACAmean ((F(2,12) = 5.02, p=0.03)) and Oxy‐Hb ((F(2,12) = 5.86, p= 0.02)). Post hoc test revealed that a voluntary start to hand grip exercise increased ACAmean by 2.6±1.0 cm/s (p=0.002) v.s. 0.7± 2.0 cm/s for a cued start (p=0.3) and increased Oxy‐Hb by 11.6±10.7 µM (p=0.02) v.s. 2.9±9.5 µM for a cued start (p=0.7) at the onset of exercise when compared to baseline. Furthermore, there was tendency in ACAvci to also increase with voluntary start by 3.9%±6.0 (p=0.06) vs. cued start 0.43% ± 6.0 p=0.57 from baseline to the initial onset of exercise. These primary findings suggest that moderate dynamic hand grip exercise elicits similar anterior cerebral blood flow and prefrontal oxygenation responses previously observed and thus, showing similar metabolic demands as finger‐tapping.
Monensin and its analogues show anti‐glioblastoma activity in an organoid model of cancer
Authors: Urbaniak Alicja , Reed Megan R. , Heflin Billie , Gaydos John , Piña‐Oviedo Sergio , Jędrzejczyk Marta , Klejborowska Greta , Stępczyńska Natalia , Chambers Timothy C. , Tackett Alan J. , Rodriguez Analiz , Huczyński Adam , Eoff Robert L. , MacNicol Angus M.
Published: May 2022

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite numerous clinical trials, the standard of care therapy has remained unchanged for the last decade. One of the main problems contributing to the limited effective treatment options and poor overall survival is the lack of models which can reliably recapitulate tumor heterogeneity. Organoids are 3D self‐organized structures which mimic tumor architecture, microenvironment, and cellular interactions which makes them an improved model for anti‐cancer drug discovery.

Monensin (MON) is a polyether ionophore antibiotic characterized by wide range of biological properties including anti‐cancer activity. In order to identify more potent compounds based on the scaffold of MON, we investigated the anti‐GBM activity of 14 novel esters and urethanes of MON. In 3D mini‐ring cell viability assays, we identified seven analogues (IC50 = 91.5 ± 54.4 ‐ 291.7 ± 68.8 nM) more potent towards GBM than the parental MON (IC50 = 612.6 ± 184.4 nM). These analogues induced DNA fragmentation in an organoid model of GBM, suggestive of apoptotic cell death. Furthermore, the most potent analog, compound 1, significantly reduced GBM cell migration, induced PARP cleavage and degradation, increased ɣH2AX signaling and increased expression of the autophagy marker LCII. To investigate the activity of these novel compounds in a tumor microenvironment, we have developed a host:tumor hybrid 3D organoid system. For host tissue, we generated human cerebral organoids (COs) from hiPSCs. The COs displayed multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1+), primitive ventricular system (SOX2+/Ki67+), intermediate zone (TBR2+) and cortical plate (MAP2+). These findings suggest the level of differentiation and development of our COs as equivalent to early stage human fetal brain. We then co‐cultured RFP‐labeled U87MG cells with fully formed COs. After establishing U87MG tumor formation, hybrid organoids were treated with MON or compound 1. Compound 1 significantly reduced U87MG tumor size after four days of treatment. Our findings highlight the therapeutic potential of MON analogues towards GBM and support further research and clinical development of these compounds.
Mechanical Properties of Embryonic Mouse Cartilage and Bone
Authors: Gifford‐Mahany Julian A. , Kiemle Sarah , Kawasaki Mizuho , Tighe Timothy , Kawasaki Kazuhiko , Richtsmeier Joan
Published: May 2022

Differences in material characteristics of cartilage and bone are primarily known through investigation of postnatal specimens but the mechanical properties of these tissue during embryogenesis are less well studied. Importantly, embryonic cartilage and bone have not been tested extensively in aqueous environments, limiting the validity of previous investigations. The purpose of this project is to characterize the stiffness and strength of embryonic cranial bone and cartilage using atomic force microscopy (AFM). We test the hypothesis that embryonic cartilage is stronger than embryonic bone against the null hypothesis that embryonic tissue response will be comparable to adult tissue in which bone is the stronger of the two tissues. In vivo conditions are simulated by immersing tissues in room‐temperature phosphate‐buffered saline before and during testing. Data are acquired using calibrated AFM probes which yield quantitative and qualitative data. Force‐distance curves include data on topography, adhesion, and DMTModulus, the latter of which can be transformed into Young’s Modulus through models which approximate the shape of the AFM probe, including the Sneddon model. Calcified tissue intended for testing is verified on the micro scale using AFM topography and on the macro scale using calcein dye. Each 2 square micron topographical image is accompanied by 16,384 force versus distance curves which yield a value for modulus of elasticity. Data show DMTModulus values ranging from approximately 200 kPa to 7.7 MPa for embryonic cartilage and 120 kPa to 3.0 MPa for embryonic bone providing preliminary data indicating that cartilage varies more than bone and may be significantly stronger than bone in mouse embryos.
Structure of a Therapeutic Antibody in Complex with MUC16 Reveals a Conformational Epitope Influenced by Antigen Glycosylation
Authors: Brooks Cory L. , Aguilar Eric N. , White Brandy , van Faassen Henk , Michaud Sarah , Goodlett David , Brooks Teresa , Hussack Greg , Henry Kevin , Radhakrishnan Prakash
Published: May 2022

The integral membrane glycoprotein Mucin‐16 (MUC16) has emerged as an important cancer antigen that displays a high degree of tumor selectivity. MUC16 is often overexpressed and involved in tumorigenesis in several malignancies, including pancreatic and ovarian cancer. The role of MUC16 in cancer progression is complex and provides multiple points for therapeutic intervention. However, despite clinical interest in MUC16 as an immunotherapy target, surprisingly little is known regarding how antibodies bind the protein. Here we report the humanization, epitope mapping, and structure determination of a MUC16 specific therapeutic antibody. The antibody was humanized using a germline complementary determining region (CDR) loop grafting approach and produced by transient transfection in Chinese hamster ovary (CHO) cells. The humanized and murine antibodies displayed nearly identical binding affinity to a recombinant MUC16 SEA (Sperm protein, Enterokinase, and Agrin) domain as measured by enzyme linked‐immunosorbent assay (ELISA) and surface plasmon resonance (SPR). High‐resolution x‐ray structures of the antibodies indicated no significant changes associated with humanization. Initial epitope mapping using an ELISA and overlapping MUC16 constructs suggested that the antibody epitope was localized to a SEA domain and was non‐linear and conformational in nature. A more detailed epitope mapping study carried out using hydrogen‐deuterium exchange mass‐spectrometry revealed five regions on the SEA domain that resulted in reduced deuteration when the antibody was bound to the antigen. These results were confirmed by x‐ray structures of the murine and humanized antibodies complex with the SEA domain. The structures revealed a complex, non‐linear structural epitope with a long b‐hairpin forming the center of the interaction. Finally, a fully glycosylated recombinant SEA domain was produced by transient transfection in CHO cells and used to assess the role of MUC16 glycosylation on antibody binding. Surprisingly, the densely glycosylated SEA domain bound the antibody with approximately 2‐fold higher affinity compared to the unglycosylated domain, suggesting a role for antigen glycosylation in mediating antibody binding. The results presented here represent the first structural characterization of an antibody in complex with MUC16 and reveal a complex, non‐linear epitope influenced by glycosylation. These findings will help to accelerate the clinical development of this promising therapeutic agent.
The Impact of Secondary Bacterial Infections in COVID‐19 Patients on Mortality and the Immune Response in the State of Arizona
Authors: Potter Pamela , Jiaxin Bai , Villait Akash , Khin Brian , Vu Cathy , Ihms Benjamin , Krzeczowski Rachel , Ashurst John , Santarelli Anthony , Smith Victoria
Published: May 2022

The literature on COVID‐19 has focused primarily on urban centers and suggest an increased risk of mortality in COVID‐19 patients coinfected with bacteria compared to those solely infected with COVID‐19. In patients screened or admitted to suburban and rural hospitals in Arizona, mortality increased with co‐infection. The impact of bacterial coinfections on the immune response was studied by comparing white blood cell (WBC) and lymphocyte counts as well as the relationship between duration of steroid use and mortality in coinfected COVID‐19 patients investigated.

Methods

A chart review of 677 SARS‐CoV‐2 patients was conducted at during December 2020 to March 2021. Patients solely infected with COVID‐19 (COVID‐19 patients) were compared to patients with concurrent bacterial or fungal infections (coinfected patients). Parameters included admission duration, ICU duration, mortality, WBC, lymphocytes, and duration of steroid use. WBC and lymphocytes were compared amongst COVID‐19 and coinfected patients that survived or that expired during their hospital stay. COVID‐19 positive patients not admitted to hospital were used as controls.

Results

The admitted and ICU duration of coinfected patients was significantly (p < 0.05) higher (12.93 vs 10.92 days) than COVID‐19 patients (6.39 vs 4.38 days), as was mortality rate (22.5% vs 9.8%). WBC was higher (9.4x103) in coinfected patients than COVID‐19 alone (7.9x103). Lymphocytes were lower (2.03x103) in coinfected patients compared to COVID‐19 alone (2.41x103). A significant difference in WBC was seen between expired COVID‐19 patients (10.1x103), expired coinfected patients (10.01x103) and COVID‐19 non‐admitted patients (6.44x103). Lymphocytes were lower in surviving coinfected patients (I.96x103) and expired COVID‐19 patients (I.61x103) than COVID‐19 non‐admitted patients (7.33x 103). Expired coinfected patients were treated with steroids for a longer average duration (9.22 days) when compared to expired COVID‐19 (6.77 days).

Conclusion

The presence of other bacterial infections increased hospitalization and mortality rate of patients admitted to a suburban hospital with COVID‐19 infection. Patients with co‐infections needed longer steroid treatment, had higher WBCs but lower lymphocytes. Thus, co‐infection presents significant challenges for treatment of patients with a diagnosis of COVID‐19.
Endothelial Inwardly Rectifying K+ Channel Subunit 2.1 Critically Enables Flow‐mediated Dilation in Cerebral Resistance Arteries
Authors: Kowalewska Paulina M. , Sancho Maria , Fabris Sergio , Welsh Donald G.
Published: May 2022

Cerebral blood flow is a highly regulated process tied to the expression of ion channels in endothelial and smooth muscle cells. Of particular note are endothelial inwardly rectifying K+ channels (KIR), membrane proteins that presumably sense hemodynamic stimuli such as blood flow. This study sought to clearly define the role of endothelial KIR2.1 in cerebral arterial blood flow sensing using floxed control and endothelial KIR2.1‐/‐ mice. Experiments progressed from single cell (patch‐clamp electrophysiology of cerebral endothelial and smooth muscle), to isolated vessel (myography of cerebral resistance arteries) and finally to intact organism (arterial spin‐labeling magnetic resonance imaging). Electrophysiological analysis confirmed the presence of KIR activity in endothelial cells from floxed but not endothelial KIR2.1‐/‐ mice; the former was subsequently shown to be flow sensitive. Smooth muscle KIR activity was comparable among the two animal groups. Vessel experiments next revealed that flow‐induced vasodilation was diminished in endothelial KIR2.1‐/‐ mice. In the absence of endothelium, Ba2+‐induced constriction was comparable among the two groups consistent with preserved smooth muscle KIR activity. Intriguingly, the diminishment of cellular and tissue level responses in endothelial KIR2.1‐/‐ mice did not lead to apparent cerebral flow abnormalities (cortex, cerebral nuclei, hippocampus, thalamus, hypothalamus and midbrain), at baseline or following a systemic blood pressure challenge (intraperitoneal phenylephrine). These findings underscore a role for endothelial KIR2.1 activity in hemodynamic sensing, one that is subtle and dynamically balanced with other mechanisms that set cerebral arterial blood flow.
The Role of Skeletal Muscle Intrinsic Clock in the Development and Progression of Cancer Cachexia
Authors: Lin Jason , Counts‐Franch Brittany , Powell Zereque , Ramanathan Chidambaram , Carson James , Puppa Melissa
Published: May 2022

Cancer cachexia is a chronic wasting disorder characterized by severe loss of skeletal muscle that affects approximately 80% of cancer patients, accounting for about 20 – 30% of cancer‐related deaths. Altered rest‐activity is a hallmark of circadian disruption and is often seen in cachectic patients. Additionally, loss of skeletal muscle‐specific clock gene results in muscle atrophy similar to that seen in cancer cachexia. However, it is unknown how skeletal muscle clock gene expression is altered with the progression of cachexia. Therefore, we investigated skeletal muscle clock gene expression during cachexia. ApcMin/+mice (MIN) were harvested in the morning and evening and were analyzed for clock gene expression. There was no effect of cancer on muscle Bmal1 (p = 0.55) and Clock (p = 0.59) expression. Cry1 expression showed no significant difference compared to BL – 6 mice (p = 0.24). Per 2 (p = 0.07) and Per 3 (p = 0.02) increased expression in the evening; however there was no difference between MIN and BL – 6 (Per 2 p=0.97, Per 3 p=0.87). Rev‐ERBα showed decreased expression in BL – 6 mice in the evening (p<0.001). However, there was no difference between the morning and evening MIN mice (p=0.31). RORα showed increased expression in BL – 6 mice in the evening (p = 0.03). Additionally, BL – 6 evening was significantly higher compared to MIN evening (p=0.01). However, there was no difference between the morning and evening MIN RORα expression (p>0.99). In LLC conditioned media treated myotubes, myotube diameter was decreased (p<0.001), and Bmal1 expression was decreased after 4 days in LLC conditioned media. In conclusion, the data suggest circadian clock expression is altered in a cachectic environment. Addition research is needed to understand the influence of these changes on the progression of cancer cachexia. Understanding the role of muscle‐specific circadian clock could lead to potential therapeutic interventions to attenuate cancer cachexia.
Thermodynamic analysis of SL1 1x2 internal loop in SARS‐CoV‐2
Authors: Eaheart Quinn A. , Grover Neena
Published: May 2022

SARS‐CoV‐2 utilizes a positive‐sense single stranded RNA as its genomic material. This single stranded mRNA hijacks the host cell’s ribosome to translate its own proteins. Upon infection of a host cell, SARS‐CoV‐2 produces polyprotein 1ab which is then cleaved into 16 non‐structural proteins. Non‐structural protein 1 (Nsp1) inhibits host cell translation by binding to the 40S ribosomal subunit. Stem loop 1(SL1) in the 5’‐UTR of the mRNA mediates the binding of Nsp1 to the ribosome. SARS‐CoV‐2 specifically contains a 1x2 internal loop within the SL1 that is not seen in other coronaviruses. In this study, we are examining the thermodynamic properties of the 1x2 internal loop of SL1. UV‐visible thermal melts and isothermal titration calorimetry (ITC) were performed on various DNA and RNA constructs containing the 1x2 internal loop in SL1 in the presence of 1 M KCl or 10 mM magnesium chloride at two different pHs. Initial results show additional base pairing at lower pH in 1 M KCl buffer, indicating the formation of an A+•C base pair.