bluuebunny/crossref_metadata_2025 huggingface dataset:
β‐Caryophyllene ameliorates cisplatin‐induced nephrotoxicity in a cannabinoid 2 receptor‐dependent manner
Authors: PACHER PAL , Horvath Bela , Mukhopadhyay Partha , Wink David , Gertsch Jürg
Published: June 2021

(E)‐β‐caryophyllene (BCP) is a natural sesquiterpene found in many essential oils of spice (best known for contributing to the spiciness of black pepper) and food plants with recognized anti‐inflammatory properties. Recently it was shown that BCP is a natural agonist of endogenous cannabinoid 2 (CB(2)) receptors, which are expressed in immune cells and mediate anti‐inflammatory effects. In this study we aimed to test the effects of BCP in a clinically relevant murine model of nephropathy in which the tubular injury is largely dependent on inflammation and oxidative/nitrative stress. β‐caryophyllene dose‐dependently ameliorated cisplatin‐induced kidney dysfunction, morphological damage, and renal inflammatory response (chemokines MCP‐1 and MIP‐2, cytokines TNF‐α and IL‐1β, adhesion molecule ICAM‐1, and neutrophil and macrophage infiltration). It also markedly mitigated oxidative/nitrative stress and cell death. The protective effects of BCP against biochemical and histological markers of nephropathy were absent in CB(2) knockout mice. Thus, BCP may be an excellent therapeutic agent to prevent cisplatin‐induced nephrotoxicity through a CB(2) receptor‐dependent pathway. Given the excellent safety profile of BCP in humans it has tremendous therapeutic potential in a multitude of diseases associated with inflammation and oxidative stress.
Calcineurin β Regulates NADPH oxidases in Kidney Hypertrophy
Authors: Williams Clintoria R. , Gooch Jennifer L.
Published: June 2021

Renal failure occurs when kidneys are unable to appropriately adapt to stress. Hypertrophy, an adaptive response, enables an organ to meet functional demands. We reported that calcineurin (Cn) is required for kidney hypertrophy. Since studies have also implicated NADPH oxidases (Nox) in kidney changes, we hypothesize that Nox and Cn cooperate in a common signaling pathway. We tested this hypothesis in vitro using primary kidney cells lacking a catalytic subunit of Cn (CnAα−/− or CnAβ−/) and in vivo using WT and CnAβ−/− mice. In vitro, hypertrophic factors stimulate Cn activity in WT and CnAα−/− cells but fail to induce activity in CnAβ−/− cells. Furthermore, CnAβ over‐expression induces hypertrophy in WT cells; while, CnAβ−/− cells fail to develop hypertrophy, indicating that CnAβ is required for hypertrophy. Interestingly, CnAβ−/− cells generate less oxidative stress, suggesting that CnAβ‐mediated hypertrophy may involve Nox. Loss of CnAβ reduces expression of oxidative stress‐generating enzymes Nox2 and Nox4. Inhibition of NFAT, a CnAβ‐regulated transcription factor, decreases Nox2 and Nox4 expression, suggesting that the CnAβ‐NFAT pathway modulates Nox. In vivo, diabetic CnAβ−/− mice have diminished renal hypertrophy and less upregulation of Nox4. These data reveal that CnAβ‐NFAT regulates Nox expression and plays an important role in hypertrophy.
Chronic treatment of spontaneously hypertensive rats (SHR) with the apocynin prevented the development of hypertension and endothelial dysfunction, but increased the effect to phenylephrine.
Authors: Perassa Ligia Arnedo , Potje Simone Regina , Lima Mariana Silva , Graton Murilo Eduardo , Pernomian Laena , Bendhack Lusiane Maria , Antoniali Cristina
Published: June 2021

We hypothesized that apocynin (APO), a NOX inhibitor, prevents the development of hypertension and endothelial dysfunction in SHR. SHR was treated from the 4th to 10th week of life with APO (30 mg/Kg/day, in drinking water). At 10th week, MAP, in vivo ACh (2μg/Kg) and phenylephrine (Phe, 8μg/Kg) effects and endothelial cells (EC) reactive oxygen species [ROS] and nitric oxide [NO] concentrations were evaluated. EC were removed from the thoracic aorta and incubated with DHE, a fluorescent dye to ROS, or DAF‐2 DA, to NO. Fluorescence intensity (U) was recorded in EC, before and after acetylcholine (1μmol/L) stimulation. The results (mean±SEM) were compared between SHR+APO, SHR and Wistar (W) rats (ANOVA, p<0.05). MAP was lower in SHR+APO (129.5± 2 mmHg; n=9) than in SHR (160.3± 3). ACh effect was reduced in SHR (−22.2 ± 2 mmHg × W: −31.1± 2), but it was increased in SHR+APO (−40.5 ± 3). Phe effect was increased in SHR+APO (+50.9±1 mmHg × SHR: +42.7±2 × W: +41.8±1). Higher [ROS] was observed in SHR EC (2880.0±0.2U × W: 1822.0±0.1), but it was reduced in SHR+APO (2090.0±0.2). [NO] was increased in SHR+APO (3368.0±0.3 U) and W (3180.0±0.2 × SHR: 2057.0±0.1). ACh increased [NO], and it was higher in SHR+APO (6320.0±0.3 × W: 4526.0±0.3 × SHR:2259.0±0.1U). We demonstrated that the chronic treatment with APO prevented the development of hypertension and endothelial dysfunction in SHR. However, APO increased Phe effect in SHR.
Vulnerability of the neonatal respiratory control system to sustained hypoxia
Authors: MacFarlane Peter Mathew , Mayer Catherine Ann
Published: June 2021

The developing respiratory neural control system is particularly vulnerable to environmental challenges including changes in oxygenation. In the current study, we investigated how neonatal hypoxia exposure during selected periods of development modifies the ventilatory response to acute hypoxia (hypoxic ventilatory response, HVR). Using whole body plethysmography, the magnitude of the acute HVR was measured in neonatal rats following exposure to sustained hypoxia (SH, 11% O2) during selected periods of development. Neonatal rats were exposed to SH between postnatal (P) age P1–5, P10–15, or P20–25. The magnitude of the acute HVR in rats exposed to SH between P1–5 (40.4±8.7% of baseline) and P20–25 (55.5±9.5%) was similar to age‐matched normoxic rats (35.9±3.6% and 49.6±6.0%, respectively). In contrast, the HVR was absent (4.4±11.1%) in rats exposed to SH between P10–15, revealing a vulnerable period of development in which the respiratory control system mediating the HVR is disturbed by SH. These data may be important to our understanding of the importance of avoiding prolonged periods of reduced O2 saturation in the intensive care of preterm infants during uniquely sensitive periods of development. Funding: Dept. of Pediatrics, Rainbow Babies and Children's Hospital.
Effects of exposure do air pollution on placental structure
Authors: de Fatima Soto Sonia , Lopes Karen , Veras Mariana Matera , Heimann Joel
Published: June 2021

Objective

To evaluate the effects of exposure to air pollution during the pre‐gestational or gestational period.

Methods

Female Wistar rats (7–9/group) were exposed to filtrated (F) or polluted (P) air in a concentrator of atmospheric particles for 3 weeks. At 12 weeks, after mating, rats were divided in 4 groups exposed to F or P (FF, FP, PF, PP). At 19th day of pregnancy: fetal placenta (Pl), decidua (Dc) and fetus (F) mass; Pl and Dc longitudinal (LD), transversal (TD) diameters and thickness (T); Pl, Dc and serum ACE activity; serum Aldosterone were measured.

Results (mean±SEM – p<0.05):

FF
FP
PF
PP

Mass (g)
Pl+Dc
0.63±0.010
0.61±0.009
0.57±0.0141
0.55±0.0071

Pl
0.42±0.006
0.44±0.006
0.40±0.0052
0.43±0.004

Dc
0.13±0.005
0.14±0.008
0.12±0.0051
0.12±0.0071

Diameters ratio (LD/TD)
Pl
1.08±0.02
1.24±0.043
1.11±0.02
1.17±0.02

Dc
1.07±0.02
1.16±0.03
1.10±0.03
1.25±0.043

T (mm)
Pl
4.42±0.10
4.16±0.06
3.93±0.094
4.40±0.11

Dc
3.15±0.10
2.77±0.095
2.92±0.09
2.95±0.11

ACE (nmol His‐leu/min/mL)
Pl
1.05±0.07
0.99±0.11
1.12±0.10
1.35±0.24

Dc
0.83±0.07
0.67±0.06
0.59±0.055
0.53±0.055

Serum
33.50±1.39
28.89±1.375
28.90±1.675
26.53±1.065

vs FF, FP;

vs FF, FP, PP;

vs FF, PF;

vs FF, PP;

vs FF.

Serum Aldosterone was similar among all experimental groups.

Conclusions

Exposure to air pollution affects placental structure indicating a possible prejudice of fetal nutrition.

Supported by FAPESP and CNPq.
Novel use of a three‐dimensional in vitro environment; does flexor and extensor tendon cell behavior differ?
Authors: Stanley Rachael Louise , Carolin Amy Miriam , Agabalyan Natasha Anahid , Evans Darrell John Rhys
Published: June 2021

Flexor tendons commonly suffer strain‐related injuries that are challenging to treat. Extensor tendons rarely suffer such damage. Investigating the differences between these tendon types may help us develop new treatments. Traditional 2‐dimensional culture of tendon cells does not appear to adequately replicate the 3D structure of tendons; therefore we investigated the differences of chick flexor and extensor tendon cell behavior in 2D and 3D environments. By creating cylindrical collagen gel structures seeded with separate cell types, we examined cell morphology and extracellular matrix (ECM) protein expression. Results demonstrated that in a 3D environment, both cell types and ECM proteins were arranged in the direction of tension; a pattern similar to tendons in vivo. Our results show that use of a 3D gel construct is an easy and reproducible method for future in vitro tendon research. To our knowledge, this is the first study to compare flexor and extensor cells in a 3D environment. Funded by The Leverhulme Trust, The Wellcome Trust and Brighton and Sussex Medical School.

Grant Funding Source: N/A
Structural and functional evolution of epithelia and cell‐cell adhesion complexes
Authors: Nelson W. James
Published: June 2021

A tube surrounded by a layer of cells, called a simple epithelium, is the most basic tissue organization in animals. Epithelial organization, which requires cadherin‐catenin cell adhesion complexes that glue cells together and organize the interior cytoskeleton of the cells, is thought to have evolved at the time animals first appeared, about 600 million years ago. I will show that an epithelial tube occurs in organisms that are much more primitive than animals, and that the mechanisms involved are similar to those in animals. Detailed biochemical analysis of cadherin‐catenin complexes during evolution reveals variations in mechanisms regulating cell‐cell adhesion and actin cytoskeleton interactions that may be tailored to specific functional requirements in different systems.
Propranolol stimulates angiogenesis during healing of ischemic wounds
Authors: Monte‐Alto‐Costa Andrea , Bandeira Ana Carla Balthar
Published: June 2021

The aim of this study was to evaluate the response to different doses of propranolol during cutaneous ischemic wound in rodents. Wistar rats were treated with propranolol at concentrations of 3 and 6 mg/kg/day, control group received only vehicle. Propranolol administration began on the day of injury, and was administered daily until euthanasia. Bilateral incisions were performed the dorsum of each animal. The flap was sutured and a full‐thickness wound was made between incisional lesions Propranolol treated with 3 mg group showed a reduction in total wound area when compared to control group. Also, propranolol treated with 3 mg group showed an increased of density of blood vessels, highest PECAM‐1 expression and highest blood perfusion in ischemic skin and in the wound bed when compared to control group. In summary, we suggest that propranolol administered at 3 mg/day was able to modulate angiogenesis and improved healing in this rodent model.
The evolution of avian retinal morphology and its relationship with locomotor head movements and foraging
Authors: Hancock Jennifer Ann , Biknevicius Audrone R.
Published: June 2021

In order to understand the evolution of avian retinal morphology, we collected known data on the number and location of the area centralis and foveae in 165 species of birds. From these data, 10 distinct retinal patterns were found: nasal unifoveate, with either a circular area or band‐shaped area; bifoveate with circular areas or a band‐shaped area; temporal unifoveate with a circular area; no area centralis; afoveate with a circular nasal area; afoveate with a circular temporal area; temporal unifoveate with a band‐shaped area; and afoveate with a band‐shaped area. The retinal patterns were mapped onto a phylogeny, and the ancestral character state for birds was found to be a nasal unifoveate retina with a band‐shaped area. The evolution of the other 9 retinal patterns appears complex, involving retentions, directional changes toward different configurations and reversals. Data was also collected on foraging behavior and locomotor head movements. Their relationships to retinal morphology were analyzed using pairwise comparisons. None of the pairwise comparisons showed significant relationships; however, some trends were apparent both within clades and across the phylogeny. One surprising result was that head‐bobbing behaviors were not limited to birds with a specific retinal morphology, as it occurred in afoveate, unifoveate and bifoveate birds.
A Century of Body Snatching: A History of Cadaver‐ Acquisition in Kingston, Ontario from 1820–1920
Authors: Belyea Scott , Duffin Jacalyn , Easteal Ron
Published: June 2021

Practical anatomy has always required bodies for dissection, and, when legal avenues failed to supply cadavers, more illicit practices developed. Scholarly and popular works on body snatching have addressed grave robbing in the United Kingdom and North America. The rise of American medical schools and their reliance on grave robbing have been well‐documented through substantial works by Suzanne Shultz and Michael Sappol, while the Canadian landscape has yet to be researched as thoroughly. This work begins to fill the gap by focussing on grave robbing in late nineteenth‐century Eastern Ontario. Lying between Toronto, and Montreal, and in close proximity to the American border, the area surrounds Kingston's medical school, founded in 1854. Sources include newspapers, pertinent legislation, and archival records of the Medical Faculty and hospital. They identify dozens of local body‐snatching cases with an uneven distribution through time, and they trace the public and medical reactions to grave robbing for anatomical study. Factors included the shortcomings of legal avenues of cadaver‐acquisition, unclaimed remains in the Kingston Penitentiary, and the city's cemetery practices. A more complete view emerges of early Canadian medical life and a reconsideration of the previously‐suggested grave‐robbing landscape in Canada with its North American implications.
Characterization of Acinetobacter baumannii umuDC
Authors: Nguyen Linda , Norton Matthew , Godoy Veronica
Published: June 2021

One reason for the rise of bacterial antibiotic resistance is acquisition of new mutations through DNA damage. Lesions on the DNA obstruct regular DNA replication polymerases. Upon DNA damage, cells induce the DNA damage response; one effect is an increase in DNA damage tolerance because they recruit Y family DNA polymerases with active sites large enough to bypass lesions. However, Y family DNA polymerases have low fidelity and may lead to errors in replication and thus mutations, some of which may result in antibiotic resistance. In Escherichia coli, there are two such polymerases, Pol IV and Pol V, the latter being the most error prone. In Acinetobacter baumannii there are multiple genes encoding DNA Pol V, and these are organized as operons (as well as unlinked genes). The multiple copies may be active in A. baumannii, and the activities of the products they encode may be comparable to their counterparts in E. coli. The operons and unlinked genes coding for Pol V in A. baumannii will be characterized to determine if their activities differ to the single operon in E. coli.
Definitive evidence using enucleated cytoplasts for nongenomic effects of STAT5a/b siRNAs on ER structure
Authors: Sehgal Pravin B , Lee Jason E , Yang Yang‐Ming , Yuan Huijuan
Published: June 2021

STAT5a/b species are well‐known as transcription factors that regulate nuclear gene expression. However, we observed that STAT5a associated with the Golgi apparatus in HPAECs and that siRNA‐mediated knockdown of STAT5a/b led to the rapid development of a dramatic cystic change in the endoplasmic reticulum (ER) characterized by deposition along cyst membranes and tubule‐to‐cyst boundaries of reticulon‐4 (RTN4/Nogo‐B), atlastin‐3 (ATL3) and Golgi fragmentation. Moreover, there was marked accumulation of the 63kDa cytoskeleton‐linking membrane protein and ER‐spacer CLIMP63/CKAP4 within the cysts. That the STAT5a/b‐siRNA‐induced cystic ER phenotype developed in the presence of a transcription inhibitor DRB had suggested that the mechanism was nongenomic. We have now definitively tested the requirement for the nucleus in eliciting the STAT5a/b‐siRNA‐induced cystic ER phenotype. Enucleated HPAEC cytoplasts were prepared using adherent 35 mm cultures using the cytochalasin B‐centrifugation method (typically yielding 65–75% enucleation). STAT5a/b siRNAs readily elicited the cystic ER phenotype including the marked luminal accumulation of CLIMP63 and Golgi fragmentation in the recovered HPAEC cytoplasts demonstrably lacking a nucleus. These studies provide unequivocal evidence for a nongenomic mechanism underlying the cystic change in ER structure elicited by STAT5a/b knockdown.
Molecular Basis of Histone Acetyllysine Recognition by the BRPF1 Bromodomain
Authors: Glass Karen Champagne , Poplawski Amanda , Hu Kaifeng , Lee Woonghee , Peng Danni , Carlson Samuel , Shi Xiaobing , Westler Milo
Published: June 2021

The monocytic leukemic zinc‐finger (MOZ) histone acetyltransferase (HAT) was first identified as a fusion partner with the CREB binding protein (CBP) in a chromosomal translocation found in acute myeloid leukemia (AML). Disruption of MOZ HAT activity leads to leukemogenic transformations and oncogenesis. Bromodomain‐PHD finger protein 1 (BRPF1) is essential for MOZ HAT activity and links the MOZ catalytic subunit to the inhibitor of growth 5 (ING5) and hEaf6 subunits in the complex. BRPF1 also contains multiple epigenetic reader domains including a unique double plant homeodomain (PHD), a bromodomain (BRD) and a PWWP domain. These chromatin reader domains recognize post‐translationally modified histones and are often found in nuclear proteins that control fundamental cellular processes including gene transcription, DNA replication and recombination. Retention of these domains in leukemic fusion proteins is speculated to direct aberrant HAT activity and may be responsible for oncogenic transformations. However, the histone target(s) of the BRPF1 BRD are unknown, and its role in modulating the activity of the MOZ HAT complex is not well understood. A unique combination of peptide array, nuclear magnetic resonance, isothermal titration calorimetry and molecular docking experiments were used to identify the BRPF1 BRD ligands and characterize the molecular determinants driving histone recognition.
Histone Acetyl Transferase (HAT) HBO1 and PHD Finger Protein 17 (PHF17/JADE1) in Epithelial Cell Regeneration
Authors: Panchenko Maria , Havasi Andrea , Haegele Joseph , Bonegio Ramon , Ichimura Takaharu , Siriwardana Nirodhini , Gall Jonathan
Published: June 2021

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of chromatinal histones. We characterized the role of JADE1‐HBO1 complexes in vitro and in vivo during epithelial cell replication. Downregulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury (AKI) to examine expression of HBO1‐JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels which returned to the baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum while JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysine 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S‐HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicated JADE1‐HBO1 complex in AKI and suggested distinct roles for JADE1 isoforms during epithelial cells recovery.

NIH RO1 DK087910, and ACS IRG‐72–001‐32 (to M. V. P.). NIH 1KO8DK090143 (to A.H.).
A novel approach to detecting RNA expression in living cancer cells
Authors: Weldon Don , Ko Alex , Williams Yuko , Hsu Matthew , Johnston Grace
Published: June 2021

Elucidation of cellular regulation or disregulation can be monitored through a cells RNA expression levels. The ability to monitor gene expression within living cells that have become cancerous could provide better insight than examining fixed or lysed tissues when studying the complexities of cancerous biomarkers. However, monitoring RNA within intact cells can prove to be challenging with current techniques due to complex or harmful sample preparation techniques. Further, amplification methods can create false positives or erroneously inflate differences. Changes in RNA levels overtime are also impossible to detect without the use of duplicate samples. Here we describe the ability to detect specific RNA targets within living cells without the need for sample prep in real time. This technique also allows for the unique ability to reuse the cells following detection for additional experiments.
Prevention of ubiquitination at lysines in the N tail, R, ICL4, and NBD2 domains disrupts CFTR trafficking and immune response
Authors: Lee Seakwoo , Schiffhauer Eric , Kang Po Wei , Zeitlin Pamela
Published: June 2021

Rationale

Post‐translational modification of lysine residues in CFTR is predicted to regulate CFTR trafficking and recycling. We hypothesized that disruption of ubiquitination in lysine residues will accelerate destruction of wt CFTR and increase inflammatory responses.

Methods

Site‐directed mutagenesis, Immunoblot, confocal analysis, ELISA assay, etc.

Results

We identify seven key lysines in CFTR protein that regulate expression, maturation, poly‐ubiquitination, and trafficking of CFTR and subsequently, its role in inflammatory responses. More specifically, K14/K68/K1218 residues are important for lysosomal degradation, while the K710/K716/K1080 residues are necessary for stabilization/poly‐ubiquitination of CFTR, and the K1041 residue is critical for overall expression of CFTR. Moreover, the K710R, K716R and K710/716R mutants are inflammatory however, inhibition of lysosomal function dampens inflammatory signaling by these mutations. K14R, K68R, K1041R, K1080R and K1218R mutants are not inflammatory. Inhibition of proteasomal degradation enhances non‐inflammatory effect of K14R and K1080R mutants.

Conclusions

Post‐translational modifications in CFTR lysine residues regulate maturation and trafficking of CFTR and the inflammatory responses.

Supported by R01 HL 59410; Eudowood Division of Pediatric Respiratory Sciences
Nitrile‐synthetic enzyme involved in the formation of a carbon‐nitrogen triple bond
Authors: KOBAYASHI MICHIHIKO , NOMURA JUNPEI , HASHIMOTO YOSHITERU
Published: June 2021

We have studied microbial metabolism of nitrile [R‐CN] at the protein and gene levels. We discovered a nitrile‐synthesizing enzyme linked with nitrile hydratase (which is an industrial enzyme used for the production of amide from nitrile) in the nitrile metabolism. The enzyme, aldoxime dehydratase contains a heme as the prosthetic group, and catalyzes the dehydration of aldoxime [R‐CH=N‐OH] into nitrile [R‐CN]. Unlike the utilization of H2O2 or O2 as a mediator of the catalysis by other heme‐containing enzymes (e.g., P450), this enzyme is notable for the direct binding of a substrate to the heme ion, experimental evidence of which was obtained by means of resonance Raman analysis with an isotope technique. This enzyme is also unique in the aspect of dehydration of the substrate even in the aqueous solution. We previously reported that ferrous enzyme, containing a five‐coordinated high‐spin heme and a histidine residue as its proximal ligand, is the reactive form of the enzyme, and that another histidine in the distal heme pocket would play a crucial role in donating a proton to a substrate during aldoxime dehydration. Considering the crystal structure of the enzyme and the amino acid sequence alignment among this enzyme family, we here constructed several mutant enzymes, where each of amino acid residues is replaced by alanine by site‐directed mutagenesis. They were expressed in E. coli and purified and the properties of the resultant purified mutant enzymes were characterized. The combination of mutagenesis, estimation of the heme contents and specific activities of the mutant enzymes and spectroscopic analyses led the identification of crucial amino acid residues for the catalysis.
Apolipoprotein A5 ‐ enriched HDL is Poor Acceptor for Cholesterol in in vitro Efflux Study
Authors: Ray Assaf , Shaish Aviv , Harats Dror , Grosskopf Itamar
Published: June 2021

High density lipoprotein (HDL) plays central role in reverse cholesterol (CH) transport (RCT). Although apolipoprotein A5, a modulator of triglyceride‐rich lipoprotein metabolism, is abundant on HDL its effect on HDL‐driven RCT has not been elucidated. To test this macrophages loaded with 3H‐ CH were incubated with whole plasma, total HDL (d=1.063–1.21), HDL3 (d=1.063–1.09), preβ‐HDL (d=1.15–1.21) from apoA5 transgenic (A5tg) and from background mice. Lipoproteins from A5tg mice were associated with significantly (p<0.05) lower CH efflux (expressed as % of medium/medium + cells cpm): 54.0 ± 4.0 vs. 35 ± 2.0 in the presence of whole plasma, 19.9 ± 4.0 vs. 12.2 ± 2.0 in the presence of total HDL, 28 ± 5.0 vs. 16.0 ± 3.0 in the presence of HDL3, 8.2 ± 2.0 vs. 4.2 ± 1.0 in the presence of preβ‐HDL. When we use HDL2 (d=1.09–1.15) as acceptor, CH efflux for A5tg mice was comparable to background FVB mice. Additionally, no difference in CH efflux between the two mice strains was demonstrated in experiments that used as acceptor very low density lipoprotein (VLDL) ‐ a lipoprotein that does not involve lecithin‐cholesterol acyltransferase (LCAT) activity. While potential underlying case include differences between the groups in both lipid and apoprotein content of HDL, the lack of difference between the groups in the VLDL experiments supports the possibility that interference of apoA5 with the activity of LCAT is a likely mechanism.
Antitumoral potential ability of ATP and antimicrobial peptides against leukemic stem cells
Authors: Paredes‐Gamero Edgar J , Buri Marcus V , Ribeiro‐Filho Antonio , Barbosa Christiano MV , Nogueira‐Pedro Amanda , Miranda Antonio
Published: June 2021

Leukemia is a disease that affects the proliferation and differentiation of bone marrow cells. News therapies against leukemia stem cells (LSC) are under investigation. We have previously shown the ability of ATP (which activates P2 receptors) and antimicrobial peptides (AMPs: magainin, protegrin, polyphemusin and gomesin) to affect hematopoietic stem cells. Thus, herein explore the antitumor capability of ATP and AMPs against LSC using concentrations that do not induce cell death. Human Leukemia lineages K562 and KG1 cells were used. Immunophenotyping and cell viability were performed by flow cytometry and clonogenic assay was performed in methylcellulose. ATP promotes an increase in the percentage of LSC (CD34+CD38− Linlow/−) after three days of treatment in a concentration‐dependent manner. However, ATP leads to a decrease in the clonogenic capacity and also in the total number of K562 and KG1 cells. Similar results were obtained with the AMPs. Viability assay showed that ATP and the AMPs did not induce cell death in LSC but they were able to induce an increase of LSC population, although the clonogenic capability of this population decreased.
Modulation of late apoptotic events by ammonia in glutaminedeprived mouse hybridoma cells
Authors: Gauthier Eric R. , Abusneina Abdelmuhsen
Published: June 2021

Highly proliferating cells reprogram their metabolism in order to fulfill their requirements in biosynthetic precursors. One consequence of this metabolic adaptation is glutamine (Gln) addiction, a phenomenon whereby cells become dependent on Gln for their survival. Using Sp2/0‐Ag14 (Sp2/0) hybridoma cells, we examined the effect of ammonium ions, a product of glutaminolysis, on the viability of Gln‐deprived cells. Supplementing Gln‐deprived Sp2/0 cells with 5 mM ammonium acetate (NH4+ acetate) or 5 mM ammonium chloride (NH4+ chloride) resulted in a significant increase in cell viability. Both ammonium salts also protected Sp2/0 cells treated with the toxic Gln analog azaserine. Supplementing Gln‐starved Sp2/0 cells with either ammonium salt did not alter cytosolic cytochrome c release or caspase‐3 activation, indicating that the mechanisms triggering the apoptotic machinery remained intact. Interestingly, a significant reduction in the number of cells with apoptotic nuclear condensation and fragmentation was observed. However, neither NH4+ acetate nor NH4+ chloride interfered with DNA fragmentation and nuclear lamin A/C cleavage, two key events observed in apoptotic nuclear condensation and fragmentation. All together, these data suggest that the pro‐survival effect of ammonium salts are linked with an inhibition of late apoptotic events. Funded by NSERC‐CANADA (to ERG).
Paracellular Transport of Soybean β‐Conglycinin using a Caco‐2/HT29‐MTX Co‐Culture
Authors: Wallace Frances Lynn , Mahler Gretchen J. , Tan‐Wilson Anna , Wilson Karl A.
Published: June 2021

Soybeans are one of the eight most prevalent food allergens and the β‐conglycinins have been determined to be one of the allergenic proteins. β‐conglycinins are homo‐ or hetero‐ trimers of evolutionarily related α, α’ and β subunits. The allergenic response to seed storage proteins is thought to be due in part to the passage of undigested proteins passing through the tight junction gaps in the gut epithelium, where they are accessible to gut associated lymphoid tissue. The goal of this study is to compare the passage of homo‐ and hetero‐trimers of β‐conglycinin through a gastrointestinal epithelium model to determine whether the cell barrier will have an altered permeability to β‐conglycinins of different subunit compositions. The native β‐conglycinins were purified from soybean seeds and further separated into trimers of only α, α’, and β subunits. A Transwell plate was constructed using Caco‐2 and HT29‐MTX cells in a ratio of 3:1. The integrity of the tight junctions of the cell monolayer was gauged by measuring transepithelial resistance (TER) and occludin expression. To measure protein passage through the cell monolayer, the proteins were applied to the apical chamber and the medium collected after a period of 2 h from both the apical and basolateral chambers. The amounts of protein in both chambers were quantified using rabbit polyclonal antibodies specific to the soybean β‐conglycinins. The passage of β‐conglycinins of different subunit compositions are compared as a function of TER. The permeability of the cell monolayer can be increased by different means, such as varying the time period between cell seeding and transport experiments and chemical inhibitors. When the cell monolayer showed a TER of 250–300 ohms/cm2, there was no detectable protein transport.
The Brain‐specific BNIP‐2 homology protein Caytaxin acts as a scaffold to transport ATP Citrate Lyase on kinesin and induce neurite outgrowth
Authors: Sun Jichao , Low Boon Chuan
Published: June 2021

Mutations in the gene, ATCAY that encodes the brain‐specific protein Caytaxin, is linked to the development of the human disease, Cayman ataxia. To understand the cellular and molecular mechanisms underlying the neuronal function of Caytaxin, proteomics pull‐down was performed and ATP citrate lyase (ACL) was identified as a novel interacting partner of Caytaxin. In rat pheochromocytoma PC12 cell, Caytaxin and ACL overexpression enhanced neuronal development characterized by long protruding neurites. However, deficiency of either Caytaxin or ACL by genetic knockdown led to marked reduction in neurite outgrowth, despite the presence of the binding partners. Caytaxin requires ACL enzyme activity to promote neurite outgrowth. Further biochemical and live imaging studies revealed that Caytaxin functions as a scaffold protein that links ACL and kinesin light chain motor protein without altering the ACL activity, and that enhances ACL trafficking along the neurites. Our results therefore support the notion that Caytaxin could regulate neuronal development through its binding and trafficking of ACL. Consequently, loss of Caytaxin function could affect ACL signaling pathway, leading to ataxia.

This work is supported in part by grants from the Biomedical Research Council of Singapore and Mechanobiology Institute, co‐funded by National Research Foundation and Ministry of Education.
DNA binding and antiproliferative activity of copper (II) complexes of Quinolone‐thiazole derivatives with applications as a chemotherapeutic agent
Authors: Foreman David Rudolph , Lin Han Xing , Fox Kristin M. , Tanski Joseph M , Tyler Laurie A.
Published: June 2021

Transition metal based complexes that exhibit nuclease activity have garnered interest due to their chemotherapeutic applications. Cisplatin has been regarded as one of the most effective drugs, however toxicities and drug resistance has limited its clinical use. Copper (II) centered complexes are promising due to the importance of copper for a range of physiological processes, including enzyme and protein activity. Copper is also found in high concentrations in tumor cells as it is needed for angiogenesis. We have synthesized and characterized several copper complexes that are derived from a thiazole ligand backbone that express nuclease activity: [Cu(8OHQ(oBt))Cl2] and [Cu(8OQ)(oBt))Cl(MeOH)]. These complexes contain step‐wise structural modifications allowing structure function comparisons to be made. DNA cleavage and binding studies were carried out using gel electrophoresis, ethidium bromide, and UV‐Vis Spectroscopy. Quantitative analyses from each of the above studies were used to assess nuclease efficiency, binding efficiency and the copper compounds’ interaction with DNA and BSA.
TTP4000, a soluble fusion protein inhibitor of Receptor for Advanced Glycation End Products (RAGE) is an effective therapy in animal models of Alzheimer's disease
Authors: Zhou Bin , Rothlein Robert , Shen Jane , Valcarce Carmen , Selmer Johan , Hanhan Marwan , Kindy Mark S , Mjalli Adnan M.M. , Kostura Matthew J
Published: June 2021

RAGE is a member of the Ig supergene family that binds with nanomolar affinity to amyloid beta (Aβ) peptides which are implicated in the pathology of neurodegenerative Alzheimer's disease. RAGE expression is upregulated in Alzheimer's brains and involved in the retention of Aβ in the brain and inflammation in the neurovascular unit. Double transgenic mice that over‐express human RAGE and human amyloid precursor protein (APP) with the Swedish and London mutations in neurons develop learning defects and neuropathological abnormalities earlier than their mutant hAPP transgenic counterparts. Double transgenic mice expressing a dominant negative form of RAGE on the same mutant hAPP background, show a delayed onset of neuropathological and learning abnormalities compared to the APP transgenic background counterpart. This data suggests that interfering with RAGE signaling may have benefit in Alzheimer's disease.

To test the therapeutic benefits of inhibition RAGE signaling. We have developed a novel fusion protein, TTP4000, using the binding domains of sRAGE linked to a human Ig Fc domain. TTP4000 has similar binding characteristics to ligand as sRAGE but a superior pharmacokinetic profile. We have tested TTP4000 in a mouse model of Alzheimer's disease and show that the protein is well tolerated and efficacious. TTP4000 was shown to significantly reduce soluble Aβ levels in brains of mice. The reduction of soluble brain Aβ levels correlated with a decrease in plaque load, a reduction in inflammatory cytokine levels and improved measures of behavior in the Morris Water Maze test. These data confirm and extend previous reports implicating RAGE in the pathogenesis of Alzheimer's disease and indicate that TTP4000, an injectable RAGE antagonist, is a viable treatment for Alzheimer's disease.
The motion control of the iron‐sulfur‐protein in complex III
Authors: Esser Lothar , Zhou Fei , Yu Chang‐An , Xia Di
Published: June 2021

Bifurcated electron flow is the basis of the Q‐cycle hypothesis which describes the double efficiency of the redox chemistry of ubiquinol/ubiquinone coupled with vectorial proton transport by complex III (bc1). The separation of the two electrons in ubiquinol oxidation into two distinct paths is achieved by bridging the quinol oxidation site and the high‐potential oxidant (cytochrome c1, f) by a controlled swivel motion of the one‐electron carrier the iron‐sulfur‐ protein's extrinsic domain (ISP‐ED). While the basic mobility of the ISP‐ED has received much experimental support it is still controversial whether its motion is under a controlling influence of the substrate or a purely statistical event. Here we present structural and biochemical evidence from mitochondrial and bacterial forms of bc1 that suggests that the capture of the ISP‐ED at the quinol oxidation site is part of the mechanism and not an accidental consequence of any set of hydrogen bonds that might have formed. This evidence involves structural information from inhibited mitochondrial and bacterial bc1 with different types of inhibitors that show clear and consistent patterns of mobile or bound ISP‐ED with or without forming direct ISP‐ED‐inhibitor hydrogen bonds. Furthermore mutational data on Rhodobacter spheroides bc1 shows a variation in specific activity and kinetics that likewise points towards direct motion control of the ISP‐ED. This work was supported by the NIH intramural research program.
A novel splicing variant of rat Pregnone X receptor down regulates CYP gene expression.
Authors: Sakai Masaharu , Nakahara Sho , Yamada So , Kurata Yuya , Yamashita Yoshihumi , Hayashi Kengo , Tsutsumi Kaori
Published: June 2021

Pregnane X receptor (PXR) is a nuclear receptor that regulates transcription of both phase I and phase II drug metabolize enzymes. PXR regulate a wide range of biological and physiological processes including drug metabolism and homeostasis of numerous endobiotics. Many alternatively spliced variants of nuclear receptors have been shown to have various effects on the transactivation of the wild‐type receptor. Multiple splicing variants were also identified for the PXR mRNA. Here we report the novel splicing variant mRNA of the rat PXR (PXR‐v). PXR‐v contains 5th intron sequence in the mRNA and it translated into a c‐terminal truncated protein, which lacks a half of the ligand binding domain. This variant mRNA is predominantly expressed in the pre‐neoplastic liver. PXR‐v cDNA transfected hepatoma cells properly expressed variant mRNA and variant protein. The CYP gene, a target gene of PXR, expression was markedly down regulated in these transformants.
ABCB1 Expression in Adrenocortical Cells is Uniquely Regulated Compared to Other Cell Types
Authors: Raymond Christopher J. , Powers Jennifer L.
Published: June 2021

ABCB1 gene expression and its role in multi‐drug resistance and pharmacokinetics have been widely studied in tissues such as intestines, liver, and kidneys. The protein product, P‐glycoprotein, is believed to play a role in transport of steroids and xenobiotics. Since adrenal cells have a significant expression of this protein and also secrete steroids, we are interested in understanding ABCB1 expression in this cell type. Increases in ABCB1 expression in other cell types and species have been observed with adrenocorticotropin hormone (ACTH), steroids, activators of the steroid and xenobiotic receptor (SXR), and epinephrine. Therefore, we examined ABCB1 expression in a human adrenocortical cell line, H295R, by treating for 24 h with 10 nM ACTH, 10 μM forskolin, or 5 μM epinephrine. RNA was isolated and converted to cDNA with a commercially available kit, then analyzed using quantitative RT‐PCR. In contrast to effects reported in other cells, ACTH showed no effect on ABCB1 expression, while forksolin showed a two‐fold decrease. Epinephrine showed a less than two‐fold decrease in expression although other cell lines have shown a five‐fold increase. The differences in response may be due to differences in receptors expressed. Our previous work has shown little expression of SXR in this tissue, but significant levels of the farnesoid X receptor (FXR). Results from additional work with FXR activators will be discussed.
Control of Mitochondria Destiny by Autophagy and a Novel Mitochondrial Dynamics
Authors: Yin Xiao‐Ming , Ding Wen‐Xing
Published: June 2021

Autophagy and mitochondrial function are coupled with cellular metabolism, growth regulation and stress response. Mitochondrial quality control can be under the regulation by autophagy, in which mitochondria are subjected to autophagic degradation, i.e, mitophagy. The condition allowing mitophagy to occur is not completely understood. In a widely used mitophagy model triggered by CCCP, Parkin is required, of which the reason is not known. We found that in the absence of Parkin non‐degraded mitochondria adopted a spherical configuration, which required mitofusins. Parkin suppressed this mitochondrial dynamics by promoting mitofusin degradation. Parkin could thus block mitochondrial spheroid formation to allow mitophagy to occur. Indeed, deletion of mitofusins without concomitant expression of Parkin was sufficient to prevent mitochondrial spheroid formation and allows mitophagy to occur. Thus Parkin and mitofusins control the switch between mitophagy and mitochondrial spheroid formation. Interestingly, through both serial sections and electron tomography the three‐dimensional structure of mitochondria spheroid was found to resemble a phagosome with an internal lumen containing cytosolic materials. This mitochondrial structure thus represents a novel mitochondrial dynamics, which, together with mitophagy, could represent different strategies of mitochondrial homeostatic response.
Superoxide Scavenging by Natural Antioxidants studied using Cyclic Voltammetry
Authors: Caldwell Charles C. , Rossi Miriam , Caruso Francesco , Belli Stuart
Published: June 2021

Reactive oxygen species (ROS), including superoxide radical, are a very important area of study due to their complex involvement in cells. Among these functions is helping to generate ATP in the mitochondria, which is the main process for cells to store energy. However, in ageing, 1–2% release of ROS from the mitochondria can attack cell organelles, DNA/RNA and proteins, thus causing degenerative diseases such as Parkinson's, Alzheimer's, etc. We use cyclic voltammetry (CV), to generate the superoxide radical by reducing oxygen in DMSO solution. CV is an ideal technique as the superoxide radical is short‐lived and it is a quick method. Subsequent addition of antioxidants decreases the superoxide levels and we measure their intrinsic scavenging effects. Flavones are antioxidants found in fruits and vegetables and in our experiments; we used four flavones as scavengers, quercetin, baicalein, morin, and chrysin. These differ only in the position and number of OH groups. Using this method, we were able to establish quantitatively which flavone is a better antioxidant. Supported by HHMI 52006322.
BCL‐2 protein regulation of apoptosis in diabetic cardiomyopathy
Authors: Van Dalfsen Kelsey M , Del Gaizo Moore Victoria
Published: June 2021

Diabetic cardiomyopathy (DCM) is a complication of diabetes that affects heart function and contributes to mortality. Hyperglycemia has been identified as a major factor in DCM's development, and is believed to act in part by promoting apoptosis. Given the integral role BCL‐2 family proteins have in controlling apoptosis, the effects of hyperglycemia on BCL‐2 proteins are being studied. Using cultured cardiomyocytes and primary cells isolated from hyperglycemic chick embryos, cardiomyocyte apoptosis has been confirmed as a result of hyperglycemia. Furthermore, protein levels of various BCL‐2 family proteins have been observed to change under hyperglycemic conditions. Taken together, results from this study will contribute to the understanding of the biochemical basis of DCM and aid in the identification of potential therapeutic targets. Elon University Undergraduate Research Program, Chemistry Department, and Provost Award to VDGM.
CTBP regulates the early SEC assembly prior to onset of transcription elongation
Authors: Byun Jung S , Gardner Kevin
Published: June 2021

The C‐terminal binding proteins (CtBP1 and CtBP2) are a dimeric family of proteins encoded by two genes, CtBP1 and CtBP2 that play various roles in cell differentiation, wound healing and EMT. One member of the dimer functions to bind numerous chromatin sequence‐specific transcription factors, while the other member recruits several different chromatin modifying complexes. In this way, CtBP has a broad potential to influence the epigenetic landscape of the nucleus. CtBP shares many amino acid homology with NADH‐dependent 2 hydroxyacid dehydrogenase and previous report suggested that CtBP has a ~ 100 fold higher nanomolar affinity for NADH than NAD+ and nuclear NADH induces conformational change in CtBP. It has been known that CtBP1 physically associates with the bromodomain of histone acetyltransferase of p300 and regulates the direct interaction between the bromodomain of p300 and acetylated histones in an NADH‐dependent manner.

We found CtBP forms complexes with p300 in resting cells of HEK293ET and CtBP depletion increases early elongation complex assembly and early mRNA transcription. We also showed CtBP overexpress repressed immediate early gene expression by attenuating early elongation and CtBP depletion increased the duration of immediate early transcription.
STEP into science at Medgar Evers College, a successful strategic plan
Authors: Carroll Margaret A. , Skeete Dereck , Catapane Edward J.
Published: June 2021

STEP into Science was designed to increase the number of students earning BS degrees in Biology and Environmental Science. The program goals are to: recruit new students and non‐STEM students into Biology or Environmental Sciences; improve retention by providing academic, financial and mentoring support; foster integration of research and technology to better equip majors to be successful applicants to graduate/professional programs; and increase the number of students graduating with BS degrees. Now in our fifth year the program has had great success. We use peer recruiters to attract more high school, transfer, and non‐science college students into STEM majors and place emphasis on undergraduate research experiences to increase the quality and retention of science majors through their BS degree. Since the inception of the program, STEM enrollment more than doubled and the number of majors actively engaged in research has risen more than 90% with a concurrent increase in student research presentations at scientific conference, and an 87% increase in the number of students receiving external research internships and travel awards to attend national conferences. STEM graduates have also increased and the program anticipates that these and future STEP into Science graduates will continue on to Masters and Doctoral programs in STEM and ultimately enter rewarding careers in the science enterprise.