bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing Metabolic Changes in Cancer Cells after Treatment with an Extract Created from Walnuts
Authors: Huang Codey Y. , White J. B.
Published: May 2022

One of the main hallmarks of cancer cells is the reprogramming of energy metabolism to support the high energy and metabolite demands of constant proliferation. Although previously observed as mainly a shift from oxidative phosphorylation to glycolysis, also known as the Warburg Effect, recent research has shown that cancer cells can utilize both pathways to make energy, which has been referred to as metabolic flux. This suggests that cancer cells can adapt to their environment by reactivating or continuing to use the oxidative phosphorylation pathway to make energy. Thus, the energy metabolism of cancer cells is of therapeutic interest. The triple‐negative breast cancer cell line MDA‐MB‐231 can undergo metabolic flux, making it an ideal cell line to study the effects of metabolism‐altering drugs. Previous work from our lab showed that treatment with a Walnut Extract (WE) induced cell death in MDA‐MB‐231 cells, as well as HeLa cells. We also observed that the WE treatment targets the mitochondria by destabilizing the mitochondrial outer membrane potential, suggesting that the extract may be able to impact oxidative phosphorylation. To examine the effects on energy production, we have taken advantage of the Agilent Seahorse XFe96 Cell Metabolic Analyzer to monitor, in real time, metabolic changes in MDA‐MB‐231 and HeLa cells treated with WE. WE treatment had impacts on both glycolytic and oxidative phosphorylation processes in both cell lines. Both time‐ and dose‐dependent responses were observed, with WE treated MDA‐MB‐231 cells exhibiting a 70% reduction in basal oxygen consumption rate compared to vehicle treated cells. Additionally, basal glycolytic rate was reduced by up to 41%, and compensatory glycolysis was reduced 70%. In HeLa cells, basal oxygen consumption rate was reduced by 74%, with a 56% reduction in basal glycolytic rate and a 76% reduction in compensatory glycolysis. Quantification of ATP production rate showed an overall reduction of 73%, with a 91% reduction in mitochondrially contributed ATP production, as well as a 47% reduction in glycolytic ATP production in MDA‐MB‐231 cells. Quantitation of total ATP within the cell using a separate assay shows an 62% decrease in ATP in treated HeLa cells. We have also looked at the status of mTOR and AMPK in the cells after treatment with the WE, because of their role in energy sensing and energy production. Interestingly, our preliminary data suggests that phosphorylated mTOR levels do not change and we also do not detect any phosphorylation of AMPK, suggesting that the WE is not effecting its changes on these proteins despite the reduction in overall ATP in the cell. Taken together, our results suggest that the WE treatment can alter and reduce the rates of glycolysis and oxidative phosphorylation, and by extension, the metabolic flux of these cancer cells.
Spontaneous Intrinsic Hepatic Vascular Oscillations
Authors: Stein Paul M.
Published: May 2022

The liver is well known as a large blood reservoir that can shift both stressed and unstressed volumes passively and actively in response to changes in portal venous and hepatic arterial blood flow, inflow and outflow blood pressures, autonomic nervous stimulation, and various humoral and pathophysiological factors. Not described is the characteristic of the liver to intrinsically modulate its own vascular pressure and volume independent of extrinsic factors.

METHODS

Male Holtzman rats (n=5) were anesthetized (30 mg/kg sodium pentobarbital, i.p.) and heparinized (500 IU/kg, i.v.), the portal vein and inferior vena cava were isolated and cannulated, the liver was flushed with room temperature normal saline, and the liver quickly removed. Each liver was mounted on a scale in a 37°C chamber (95% O2‐5% CO2) and pump‐perfused (Masterflex) with donor rat blood at a baseline 1 ml/min/g of tissue. The hepatic venous pressure (Phv) was regulated by raising and lowering the blood reservoir. At each Time (see table), portal venous oscillations were counted over 3 to 12 min and the portal venous wavelet pulse pressures (Ppv) were measured over at least 40 wavelets.

RESULTS

The portal venous pressures of 3 of the 5 livers oscillated spontaneously. One lasted only 26 min while the others went on for over 200 min. The characteristics of these oscillations varied widely from animal to animal, but there usually was an irregular cycle length and uneven wavelet magnitude that tended to degrade to a diminished size with higher frequency over time (see Figure 1).

Rat
Time (Min)
Maneuver
Mean Portal Venous Pulse Pressure (mmHg)
Frequency (Wavelets/Min)
Frequency (Hz)
Cycle Length (Sec)

1
0
Baseline
2.1 ± 0.9 (SD)
8.08
0.135
7.42

95
Baseline
1.5 ± 0.8
16.10
0.268
3.73

107
Change Phv
2.5 ± 1.3
17.00
0.283
3.53

(0 to 10 mmHg)
(P<0.001 vs. 95 Min Baseline)

180
Baseline
1.3 ± 0.5
18.67
0.311
3.21

2
0
Baseline
0.8 ± 0.4
14.42
0.240
4.16

60
Baseline
0.4 ± 0.3
20.00
0.333
3.00

130
Baseline
0.4 ± 0.2
23.78
0.396
2.52

3
0
Baseline
0.6 ± 0.3
8.67
0.144
6.92

CONCLUSIONS

This is a very limited data set, providing more questions than answers, but it opens a curiosity to much more investigation to understand the function of these intrinsic hepatic vascular oscillations under normal, reflex, and pathophysiological conditions.
Identification of the Activity‐Dependent Neuroprotective Protein (ADNP) as a Novel Effector of Gαi1
Authors: Momplaisir Nathalie L. , Chandan Naincy , Lefevre Tyler , Smrcka Alan V.
Published: May 2022

The Gi family of G protein‐coupled receptors (GPCRs) drives various cellular processes to regulate physiological functions that encompass neuronal, immune, and endocrine health. Agonist‐bound GPCRS result in the activation of the heterotrimeric G protein, leading to the dissociation of the Gα subunit from the Gβγ dimer. While Gβγ has been considered to be a main driver of signaling downstream of Gi‐coupled receptors, the lack of tools to inhibit Gαi subunits without disrupting Gβγ signaling has limited our appreciation of potential novel pathways downstream of Gαi. To identify new Gαi interaction partners, our laboratory adopted an unbiased proximity‐based approach where we fused BioID2, a biotin ligase, within a specific site of the helical domain of the inactive Gαi1(Gαi‐BioID2) and the constitutively active Gαi1 (Gαi1‐Q204L‐BioID2). Using mass spectrometry, we specifically looked for targets enriched in Gαi1‐Q204L‐BioID2 samples. Among 104 targets, the activity‐dependent neuroprotective protein (ADNP) was found to be the most enriched target enriched in active Gαi1 in two separate screens. There is no previously identified relationship between Gαi and ADNP.

ADNP is a transcription factor and cytoskeletal binding protein involved in brain formation and maturation. To investigate the interaction of ADNP with active Gαi1, HEK293 cells were transfected with ADNP alone, ADNP with Gαi1‐Q204L‐BioID2, and ADNP with Gαi1‐BioID2 and supplemented with biotin for 24 hours. Cells were lysed, and streptavidin beads were used to capture biotinylated proteins. Using immunoblotting, we confirmed the selective enrichment of ADNP biotinylation by Gαi1‐Q204L‐BioID2.To further determine whether ADNP interacts with Gαi1, we used a nanoluciferase‐based complementation assay where we co‐transfected HEK cells with ADNP with Gαi1 WT and Gαi1 QL respectively. In this assay, significantly greater interaction between ADNP and Gαi1 QL was observed when compared to Gαi1 WT and ADNP.

Next, we addressed the subcellular localization of ADNP and Gαi1. Using cell fractionation, we observed that ADNP is localized to the nucleus of HEK cells while Gαi1 immunoreactivity was found in a cytosolic and membrane fraction, and in the nuclear fraction. Gαi1 expression does not alter the localization of ADNP, but ADNP can be exported and imported through the nucleus. Current work is focused on manipulation of the cellular localization of ADNP to determine where in the cell Gαi1 interacts with ADNP. Overall, we have identified ADNP as a novel interactor of active Gαi. Considering the distinct localization and expression of ADNP at different neurodevelopmental stages, further characterization of this interaction may provide new insight into Gαi‐mediated cAMP‐independent signaling pathways in neurophysiological and pathological states.
Fullerenes Materials Contribute to Ordered Interfacial Cell Water Improving Cellular Electrodynamics and Oxidative Stress Management
Authors: Bourassa Daniel J. , Tulp Orien L. , Einstein George P.
Published: May 2022

The role of water in living systems is complex and a primary mediator of localized and long‐range biological effects in the cell. Of the approximately 70% of cell water, 20‐30% forms a structured semi‐crystalline gel state of water layers, where it also hydrates surfaces of biomolecules and cell structures and is essential for life. Decreases in ordered interfacial water are associated with cellular dysfunction and disease. This interfacial water behaves quantumly. The customary Coulomb Law of electrostatics does not apply, allowing the accumulation of tissues composed of negatively charged cell bodies. Interfacial water is responsible for the thermodynamics of proton and electron transfer and long‐range quantum electrodynamic coherence coordinating quantum criticality of the cellular machinery and systemic integration necessary of complex organisms. We propose that fullerene material increases interfacial water ordering, beneficially altering its electromagnetic properties. Fullerene materials can bind with cellular structures and protein secondary structures not typically available for water interaction, through weak van der Waals forces or pi‐pi stacking arrangements containing aromatic rings with dense pi electron clouds and hydrophobic functional groups. Modulating partial charges, dipoles, and quadrupoles in fullerene materials that result from coherent microtubule‐generated electromagnetic fields helps enhance and extend long‐range ordered water layers and electrostatics between hydrophobic molecules thought necessary for the self‐assembly of bio‐macromolecules. We propose that fullerenes short and long‐range enhancement of structured interfacial water contributes to many of the observed effects of fullerenes in vivo, including a non‐stoichiometric redox mechanism for the well‐established antioxidant activity of fullerene materials.
Exploring the novel mechanistic aspects of function of a hyperthermophile two‐site exo‐amylase‐cum‐glucanotransferase displaying substrate versatility
Authors: Sarkar Arpita , Kaila Sharma Pallavi , Guptasarma Purnananda
Published: May 2022

We have recently described an exo‐amylase‐cum‐glucanotransferase, PfuAmyGT, from Pyrococcus furiosuswhich uses disproportionation to generate a pool of glucose and small malto‐oligosaccharides (MOGs) from starch, or from any single MOG (or a combination of MOGs). PfuAmyGT appears to use a combination of exo‐amylase action and excision‐and‐transfer of glucose from donor to acceptor MOGs, using a unique and novel mechanism involving two catalytically‐active sites (i.e., separate donor and acceptor sites, with a loop transferring the excised glucose) in place of the more common single site (at which the donated glucose is excised, and left, by the departing donor MOG, for being picked up by the acceptor MOG). We demonstrate that there are five residues (three glutamates and two aspartates) that are essential for activity, suggesting that different acidic side chains act at the donor and acceptor sites. We also demonstrate that PfuAmyGT acts upon substrates possessing different monomeric units and different types of glycosidic bonds (e.g., pectin, xylan or cellulose), indicating an unprecedented level of versatility, given that only one other glycosyl hydrolase (a neo‐pullulanase) has previously been reported to act upon more than one type of glycosidic bond (alpha‐1,4 and alpha‐1,6). Our bioinformatics and biochemical analyses suggest that the donor site of PfuAmyGT is processive, while the acceptor site releases and rebinds MOGs in each duty cycle. We have identified a total of six residues in the vicinity of a catalytic aspartate (D362) that potentially function to give rise to the said substrate versatility. Four of the six residues are also catalytically important for the functioning of PfuAmyGT on different substrates.
Soluble Isoform of ST2 is a Cardiac Inducer of Collagen Deposition in an Ex Vivo Model of Obesity
Authors: Corsi Romanelli Massimilano M. , Copes Chiara , Dozio Elena , Sitzia Clementina , Galliera Emanuela , Vianello Elena
Published: May 2022

Introduction

One of the main cardiac mechanosensor is the IL‐33/ST2L complex composed by the allarmin IL‐33 released into extracellular space by damaged cells during cardiac overload and its receptor, ST2L, which their binding promotes cell survival and anti‐fibrotic signaling. After damage, to stop IL‐33/ST2L signal, resident cardiac cells release sST2, the soluble isoform of transmembrane receptor of ST2, which sequesters IL‐33, functioning as a decoy receptor and blocking its signal. In obesity, the sST2 become over express especially by hypertrophic fat cells. The aim of our study is to evaluate in an ex vivo model of obesity the role of sST2 in the promotion of collagen production by cardiac cells.

Methods

Ten obese nondiabetic male Zucker rats (OB) (fa/fa‐, 10 weeks of age) and 10 lean littermates (L) (Fa/+) at the age of 25 weeks were weighted and scarified according to Italian Ministry of Health Authorization (N°325/2015PR of 2015/04/05) and their heart and visceral adipose tissue (VAT) were collected for the analysis. VAT biopsies were cut in 30mg pieces and added into a co‐culture system with H9C2 for 6‐24 and 48 hours. At each time points, cells and culture medium were collected. In all samples were evaluated ST2 by ELISA and collagen were quantified by quantitative Sircol assay for measurement of both acid‐soluble and pepsin‐soluble collagens.

Results

OB cardiac biopsies showed higher protein level of both ST2 and collagen than L rats (p<0.02 and p<0.002 respectively) and the rat weight positively correlates with ST2 cardiac protein level (p<0.05). Co‐culture data demonstrated that OB VAT promotes ST2 releases in both cardiac and fat cells, resulting an incremental increase of sST2 release in cell culture medium than L VAT in time. sST2 quantification in correspondent H9C2 co‐cultured lysate is more incremented in OB than L H9C2 after 48h.total collagen quantification in co‐culture medium resulted higher in OB than L (p<0.05).

Conclusion

Our results demonstrated in a co‐culture system that obesity promotes sST2 increase in the heart and this promotes collagen releases by cardiac cells, suggesting sST2 role in activation of pro‐fibrotic signaling and detrimental heart remodeling.
The 26S Proteasome Switches Between ATP‐Dependent and Independent Mechanisms in Response to Substrate Ubiquitination
Authors: Manfredonia Abramo , Kraut Daniel A.
Published: May 2022

The Ubiquitin‐Proteasome System is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome by the attachment of polyubiquitin chains. A number of proteins also contain ubiquitin‐independent degrons (UbID) that allow for proteasomal targeting without the need for ubiquitination. UbID substrates are degraded less processively than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust with complete degradation only occurring with relatively unstable substrates (those that were at least transiently unfolded as determined by protease sensitivity). Surprisingly, UbID degradation was unaffected by the non‐hydrolyzable ATP analog ATPγS, which halts ATP‐dependent engagement and translocation of substrates. Furthermore, ubiquitin‐dependent degradation proceeded in a strikingly similar fashion to UbID degradation in the presence of ATPγS, suggesting the 26S proteasome may have a “default” ATP‐ and ubiquitin‐independent mechanism that is capable of unfolding and degrading less‐stable proteins.
5‐HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity
Authors: Moreira Thiago S. , Shi Yingtang , Sobrinho Cleyton R. , Milla Brenda M. , Soto‐Perez Jaseph , Stornetta Daniel S. , Stornetta Ruth L. , Takakura Ana C. , Mulkey Daniel K. , Bayliss Douglas A.
Published: May 2022

A brainstem homeostatic system senses CO2/H+ to regulate ventilation, blood gases and acid‐base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2/H+‐sensitivity of RTN neurons is mediated indirectly – by raphe‐derived serotonin acting on 5‐HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT‐PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb+/Phox2b+) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These more dorsolateral Nmb+/Htr7+ neurons tended to express high levels of Nmb and low levels of the intrinsic RTN proton detectors, Gpr4 and Kcnk5. In mouse brainstem slices, CO2‐stimulated firing in RTN neurons was mostly unaffected by a 5‐HT7 receptor antagonist, SB269970 (n=11/12). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co‐injected LP‐44, a 5‐HT7 receptor agonist, but had no effect on CO2‐stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5‐HT7 receptors have negligible effects on CO2‐evoked firing activity in RTN neurons or on CO2‐stimulated breathing in mice.
Angiotensin (1‐7) exerts antioxidant effects, inhibits mTOR signaling and protects against endoplasmic reticulum stress and apoptosis in HK2 cells
Authors: Farooqui Zeba , Lokhandwala Mustafa F. , Banday Anees A.
Published: May 2022

Angiotensin (Ang) 1‐7, a bioactive heptapeptide of the renin‐angiotensin aldosterone system is known to exert antioxidant and nephroprotective effects. However, the cellular mechanisms involved in the beneficial effect of Ang 1‐7 is not entirely understood. Here, we studied the effect of Ang 1‐7 on H2O2‐mediated oxidative damage, endoplasmic reticulum (ER) stress and apoptosis in the human proximal tubular (HK2) cells and the specific mechanisms. HK2 cells were incubated with H2O2 (500 µM) and pre‐treated without and with Ang 1‐7 (100 nM). We measured reactive oxygen species (ROS) generation, ER stress, apoptosis and mTOR signaling in these cells. H2O2 treatment resulted in an increased oxidative stress in HK2 cells. Increased in the expression ER stress marker, binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), and cellular apoptosis was also observed after H2O2treatment. Moreover, H2O2 treatment resulted in the activation of mTOR complexes (mTORC1 and mTORC2) in these cells. Ang 1‐7 pre‐treatment to H2O2incubated cells significantly attenuated ROS generation, ER stress and apoptosis. These effects of Ang 1‐7 were blocked by co‐treatment with A779 (Mas receptor antagonist). Additionally, prior treatment of Ang 1‐7 inhibited the H2O2mediated mTOR activation. However, A779 treatment and transfection of HK2 cells with Mas receptor siRNA reversed the inhibitory effect of Ang 1‐7 on mTOR activities. In conclusion, our study suggests that Ang 1‐7 acting via Mas receptor could protect HK2 cells from H2O2‐induced oxidative damage through inhibition of oxidative stress, ER stress and ultimately cell apoptosis by suppressing mTOR activation.
Gut commensal Coprococcus comes diminishes the blood pressure‐lowering effect of ester angiotensin‐converting enzyme inhibitors
Authors: Yang Tao , Mei Xue , Tackie‐Yarbo Ethel , Tambari Akere Millicent , Kyoung Jun , Mell Blair , Yeo Ji‐Youn , Cheng Xi , Zubcevic Jasenka , Richards Elaine M. , Pepine Carl J. , Raizada Mohan K. , Schiefer Isaac T. , Joe Bina
Published: May 2022

Introduction

Drug resistant HTN (rHTN) affects around 15% to 20% of hypertensive (HTN) patients. The underlying mechanisms of resistance to treatment remain poorly understood. The majority of angiotensin‐converting enzyme inhibitors (ACEi) are esters, whereby we hypothesized that select gut microbiota hydrolyze ACEi rendering lower efficacy (Figure 1A). To test this hypothesis, we investigated if and which gut microbe modulates the effectiveness of ACEi.

Methods

Vancomycin, Meropenem and Omeprazole (VMO) were given to 16‐week‐old male Spontaneously Hypertensive Rats (SHR) at 50 mg/kg/day for five days. A single dose of 8mg/kg Quinapril was orally administered to both SHR and SHR+VMO, and blood pressure (BP) was recorded via radio‐telemetry. Liquid chromatography–mass spectrometry was used to measure the catabolism of quinapril. The hydrolysis of p‐nitro‐phenylbutyrate was used to measure the activity of bacterial esterase. 16S rRNA sequencing was used to study the microbial composition. At last, ester ACEi ramipril and non‐ester lisinopril were co‐administered with Coprococcus comes, respectively, to generalize the effect of C. comes on ACEi's effectiveness.

Results

Compared to the SHR, depletion of gut microbiota in the SHR+VMO group preserved the BP lowering effect of Quinapril, an ester ACEi (Figure 1B). The SHR+VMO group showed (1) reduced Coprococcus (Figure 1C); (2) lower esterase activity per gram of cecal microbiota to hydrolyze quinapril (Figure 1D); (3) a 50% lower reduction in quinapril quantity (nmol) after incubation with 1mg of cecal lysate for 3 hr (Figure 1E). C. comes,a species in Coprococcusgenus, catabolized quinapril in vitro and reduced its BP‐lowering effects in the SHR (Figure 2A‐B). Importantly, C. comes also reduced the BP‐lowering effects of ramipril (ester), but not lisinopril (non‐ester) in the SHR (Figure 2C‐D).

Conclusion

These observations constitute the first report of an unrecognized role of a select gut microbe, C. comes, in reducing the effectiveness of ester ACEi. As such, this mechanistic study serves as the foundation for expanding clinical management of antihypertensive drug resistance via manipulation of gut microbiota.
Protein engineering and biochemical/biophysical approaches for structural studies of small membrane proteins and their complexes: Application to viroporins
Authors: Majeed Saman , Ahmad Akram B. , Adetuyi Oluwatosin , Evans Eric G. , Stoll Stefan , Georgieva Elka R.
Published: May 2022

We study the structure and function of small viral membrane proteins, which are expressed in the host and function in cellular membranes. Typically, they are multi‐domain multi‐functional proteins aiding virus adaptation and proliferation. Detailed characterization of the structure‐function relationship of these proteins will help to better understand virus‐host interactions and possibly develop protein inhibitors. However, such characterization is challenging due to the proteins’ small size and dynamic nature. Our focus is on viroporins, namely the Vpu, p7 and E proteins from HIV‐1, HCV, and CoV, respectively. Viroporins form ion‐conducting oligomers in cellular membranes leading to increased membrane permeability affecting ion homeostasis in infected cells. In addition, these proteins have soluble domains involved in interactions with host proteins. Currently, the structures of full‐length viroporins are largely uncharacterized and the information about the mechanism of oligomer formation and inter‐domain communication is very limited. Here, we report our effort to produce and solve the structure of stable viroporin oligomers in lipid environment. We use protein engineering to generate a range of fusion constructs aiming to increase the protein/oligomer size and reduce the conformational heterogeneity, thus making the protein amenable for structural determination by electron microscopy (EM) techniques. We produced, expressed in E. coli and purified, several soluble constructs of Vpu, p7 and E proteins fused at their N‐terminals to maltose binding protein (MBP). Thereafter, we interacted these proteins with lyso lipid mixtures of PC and PG, and their oligomerization was observed by negative staining EM and pulse electron paramagnetic resonance (EPR). We further use size‐exclusion chromatography (SEC) to assess the heterogeneity of viroporin oligomers in lyso PC/PG. Indeed, the protein elution exhibits a relatively broad chromatographic profile, indicating the presence of more than one oligomeric form. We further use SEC to test different protein‐to‐lipid molar ratios and the effect of viroporin inhibitor binding, aiming to shift the equilibrium toward stable functional viroporin oligomers. We will also discuss results from EM studies on these stable oligomers and EPR studies on spin‐labeled viroporin assembly. The progress we made in generating protein constructs and assessing protein oligomerization and stability in lipid provides a great promise for the development of successful methodology to determine the structure of full‐length viroporin. This methodology could also be applicable to other small oligomeric membrane proteins.
Generative Deep Learning of the Single Cell Dose Response of 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin in Mouse Liver
Authors: Kana Omar Z. , Nault Rance , Zacharewski Timothy , Bhattacharya Sudin
Published: May 2022

Single cell RNA‐seq provides a new opportunity to study the heterogeneity of chemical perturbation within tissues. However, exploring the space of all cell type – chemical combinations is experimentally and financially unfeasible. This space is significantly expanded by the dose axis of chemical perturbation. Computational tools are needed to predict responses not only across tissues, but also across doses while capturing the nuances of cell type specific gene expression. Generative deep learning architectures such as autoencoders simplify the single cell expression space allowing cross‐tissue, ‐organ, ‐organism and ‐dose predictions using simple vector arithmetic. However, differing sensitivities and non‐linearities make cell type specific gene expression predictions following treatment at higher doses challenging. Here, improvements to vector arithmetic in the latent space achieve high dose cell type specific predictions better than other state of the art algorithms. We specifically look at a well‐studied toxicant, 2,3,7,8 Tetrachlorodibenzo‐p‐dioxin (TCDD), in the mouse liver as a case study. TCDD binds to and activates the aryl hydrocarbon receptor (AhR) to regulate gene expression associated with several physiological processes including xenobiotic metabolism, development, and circadian rhythm. Prediction of the effects of TCDD across liver cell types is non‐trivial as AhR is not only differentially expressed across cell types but is also differentially expressed within certain cell types along the porto‐central axis of the liver lobule. Our model predicts dose‐dependent gene expression across liver zones (i.e., portal to central hepatocytes), and across liver cell types (e.g., endothelial to stellate cells) using basal gene expression data. The model is also able to predict low dose TCDD gene expression using high dose data. Finally, using outputs from the model, an algorithm was developed to order cells based on their sensitivities to a particular toxicant by assigning a “pseudo‐dose” value to each cell. Given the model’s high performance on these tasks, the tool developed here will aid the exploration of the gene expression space of chemical perturbation.
Which Comes First: Angiogenesis or Myogenesis Following Punch Biopsy Injury?
Authors: Morton Aaron B. , Jacobsen Nicole L. , Cornelison D. D. W. , Segal Steven S.
Published: May 2022

Introduction

Skeletal muscle is integral to metabolic homeostasis, movement, and breathing. The microcirculation supplies oxygen and nutrients to myofibers while removing their metabolic byproducts. Following skeletal muscle injury, damaged myofibers and capillaries regenerate to restore tissue structure and function. However, it is unknown if angiogenesis or myogenesis is preferred when blood vessels and myofibers are removed simultaneously and permitted to regenerate. Congruent with the dependence of myofiber function and survival on their microvascular supply, we hypothesized that angiogenesis precedes myogenesis during skeletal muscle regeneration following punch biopsy.

Methods

The left gluteus maximus muscle (GM) of anesthetized male and female Cdh5‐mTmG reporter mice (age, 4‐5 months) was injured by creating a hole (diameter, 2 mm) through the muscle with a biopsy punch. Mice were studied at four timepoints: uninjured control [0 days post injury (dpi)], 7 dpi, 14 dpi, 21 dpi.

Results

Using intravital microscopy to observe the GM in anesthetized mice, regenerating microvessels grew into the biopsy zone by 7 dpi with networks traversing the residual gap by 14 dpi; nascent microvessels were perfused at 14 dpi as confirmed with fluorescent markers injected into the circulation. In contrast, myofibers were absent from the injured area at 7 dpi, began regenerating at wound edges by 14 dpi, and spanned the gap at 21 dpi. Confocal imaging and histology confirmed the presence of microvessels and absence of myofibers at 14 dpi, with both tissue components reoccupying the injured area at 21 dpi.

Conclusion

Angiogenesis precedes myogenesis during regeneration following punch biopsy of skeletal muscle.
Evolution of enzymatic function through gene duplication in the metallocarboxypeptidase family
Authors: Fajardo Daniel S. , Saint Jean Ritchie , Lyons Peter J.
Published: May 2022

Changes in the genetic code occur frequently through, for example, DNA breaks and recombination events, often leading to the duplication of genes. The M14 family of metallocarboxypeptidases suggests such a history of gene duplication. In order to understand the functional outcome of such events, we investigated more recent duplication events in the M14 family of enzymes through analysis of genomic data found in the Ensembl genome database. Of the 23 M14 genes found in most vertebrates, four genes were present as two or more copies in many fish species. The AEBP1, CPXM1, and CPZ genes were duplicated in most fish with an arrangement suggesting a large‐scale event such as whole‐genome duplication. The CPO gene was also frequently duplicated in many fish species, but with copies tandemly arranged, suggestive of crossing‐over errors. A close analysis of the synteny of four CPO paralogs found in Xenopus tropicalis suggested potential transposon‐mediated duplication. Prediction of enzyme functions for the products of these genes showed that a greater amount of neofunctionalization was present in CPO paralogs as compared with other carboxypeptidase enzymes; purifying selection to maintain function remained detectable. To further examine function, the four CPO paralogs found in Xenopus tropicalis were examined biochemically. All were expressed in HEK293T cells, but no enzymatic activity was detected. Subcellular distribution suggested a possible function on lipid droplets similar to that previously shown for human CPO. Experiments to further examine enzyme activity using the Sf9 expression system are ongoing.
Exploring The Promiscuity Potential Of 6‐Hydroxynicotinate‐3‐Monooxygenase: Consequences To Catalysis Of Adding A Nitrogen At C5 Within The Substrate’s Aromatic Ring
Authors: Belsky Sam L. , Snider Mark J.
Published: May 2022

Bacterial degradation of nicotinic acid is a model for exploring the bioremediation potential of soil bacteria. One enzyme within this catabolic pathway is 6‐hydroxynicotinate‐3‐monooxygenase (NicC), a group A flavoenzyme that catalyzes the conversion of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine. This decarboxylative hydroxylation reaction is relatively unique in biology. We recently published a study highlighting NicC’s ability to catalyze the decarboxylation and hydroxylation reaction with alternative substrates including 4‐hydroxybenzoate (4HBA) and 5‐chloro‐6‐hydroxynicotinate (5‐Cl‐6HNA). To further explore the structural basis of a substrate that enables tight binding and catalysis with NicC, 2‐hydroxypyrimidine‐5‐caboxylate (2HM5A) was characterized. This 6‐HNA analogue with an extra N atom at C5 was chosen due to its fundamental symmetry and comparable electron distribution as 5‐Cl‐6HNA. Analyses of NicC with 2HM5A show that the expected product, 2,5‐dihydroxypyrimidine (isouracil), is generated, with H2O2 occurring at a mol fraction of about 0.07, indicating that uncoupling between NADH oxidation and substrate hydroxylation is minimal. Equilibrium titration of NicC with 2HM5A by changes in A‐450 nm measure the Kd = 39 ± 6 µM, indicating that this substrate is bound more tightly than 6‐HNA (Kd= 58 ± 12 µM) but less tightly than 5‐Cl‐6‐HNA (Kd= 7 ± 2 µM). Interestingly, the overall ΔA450 elicited by the binding of 2HM5A (0.018) was smaller in comparison to the ΔA450 observed for the binding of 5‐Cl‐6HNA (0.07) or 6‐HNA (0.05). Attenuated flavin movement due the extra nitrogen of the pyrimidine ring in 2HM5A H‐bonding to the isoalloxazine ring may explain this observation. The kinetics of binding 2HM5A by NicC measured by stopped flow spectrophotometry shows that this analogue binds by a two‐step mechanism, with a nearly ten‐fold higher initial affinity for NicC prior to isomerization (ionization) than 6‐HNA. This added affinity may again be explainable by the added H‐bonding potential of this analogue. In the second step of binding, 2HM5A was observed to have a rate constant similar to 5‐Cl‐6HNA (k2= 220 s‐1), possibly indicating that increased electron density at/near the C5 position accelerates this phase of the binding mechanism. How 2HM5A affects NicC’s mechanism of flavin reduction and oxidation will also be presented. These findings provide further evidence of this monooxygenase’s potential to be engineered to hydroxylate other N‐heterocyclic aromatic compounds in bioremediation.
In vitro model of pulmonary candidiasis for testing novel therapeutics
Authors: McLaughlin Rachel M. , LaMastro Ronnie , Frazer Corey , Shukla Anita , Bennett Richard , Harrington Elizabeth , Hoffman‐Kim Diane
Published: May 2022

Invasive candidiasis is one of the most common causes of hospital‐acquired blood infections, with the most common cause being Candida albicans. These bloodstream infections often lead to long hospital stays and have a high mortality rate. Once C. albicansenters the bloodstream it can disseminate and invade many organs of the body, including the lungs, where it can inhibit function and cause further complications. Furthermore, C. albicansis commonly isolated from the lungs of cystic fibrosis patients and often causes coinfection in patients with severe COVID‐19. Current antifungal drugs exhibit high toxicity, poor water solubility, and are ineffective against some strains of C. albicans due to antifungal resistance, creating an urgent need for new antifungal therapeutics. Here, we have developed a customizable in vitroplatform to study the interaction between rat lung microvascular endothelial cells (ECs), C. albicansSC5314, and novel liposomes encapsulating the antifungal drug anidulafungin. Liposome formulations composed of hydrogenated soy phosphatidylcholine, phosphatidylglycerol, cholesterol, and α‐tocopherol were fabricated containing three different % wt/wt concentrations of anidulafungin (0.31, 1.55, and 3.10, or 1x, 5x and 10x, respectively). The minimum inhibitory concentrations (MICs) of the different liposome formulations for C. albicans were determined using a microdilution assay. After 24 hours of exposure, the MICs were found to be 12.50, 6.25, and 1.56 μg/mL for 1x, 5x, and 10x liposome formulations, respectively. Cell Counting Kit 8 was used to determine EC viability after 24‐hour treatment with the different liposome formulations. The 1x and 5x anidulafungin liposome formulations showed no significant decrease in viability up to the maximum liposome concentration tested (50 μg/mL), with the 10x formulation causing a slight but significant decrease in viability. The 1x and 5x anidulafungin liposomes were then assessed at 6.25 and 12.5 μg/mL in a co‐culture setting. C. albicans were seeded onto established EC monolayers and were allowed to form hyphae, which is the invasive form. After the two hours, the co‐cultures were treated with the antifungal liposomes. Treatment with the liposomes reduced hyphae formation and prevented C. albicansovergrowth within four hours of exposure, with the 5x anidulafungin liposome at 12.5 μg/mL showing the strongest change. These data suggest that these novel antifungal liposomes have the potential to treat pulmonary candidiasis without the toxic side effects associated with current antifungal drugs.
Quantifying Longitudinal Network Geometry Alterations in Rat Skeletal Microvasculature Subject to Ischemia
Authors: Alimorad Gabrielle , Taing Melvin , Frisbee Jefferson C. , Goldman Daniel , Kharche Sanjay R.
Published: May 2022

Study objective

Intra‐vital video microscopy (IVVM) data of rat subcutaneous skeletal muscle microvasculature under ischemic conditions at two time points (0 hours, 3 hours) was obtained from our collaborator. The objectives of this study are to elicit network geometries from the IVVM data and quantify the alterations in the network statistics of the geometries due to ischemia.

Hypothesis

We hypothesize that ischemia causes blood vessel de‐recruitment.

Methods

IVVM data from six experiments was obtained, where each experiment provided a video at the start (0 hours) and end (3 hours) of the experiment. Using consecutive still images from the videos, vessel centerlines were manually drawn using Procreate and the NIH software, ImageJ. The included vessels were chosen based on two features: (1) visible red blood cell flow and (2) clearly defined edges. The centerlines were used to guide thresholding by identifying appropriate vessels. This was followed by the generation of binarized representations of the microvascular networks. The network statistics were quantified by measuring vessel segment lengths and diameters, counting the number of bifurcations, and calculating vessel density in ImageJ.

Results

At 3 hours, the average vessel length changed from 283.91 + 252.14 pixels to 316.75 + 292.37 pixels, the average diameter changed from 16.02 + 7.99 pixels to 15.42 + 7.50 pixels, the average number of bifurcations changed from 32.42 to 18.92 and the average vessel density changed from 0.0085 + 0.0017 to 0.0061 + 0.0024.

Conclusion

There is a clear decrease in average diameter, vessel density and the number of bifurcations characterizing the microvascular networks. However, an increase in the average vessel length accompanies these findings. The data indicates that ischemia may cause de‐recruitment of blood vessels.
Pilot Study Using An Online Platform “EPrescribe” in the Training and Assessment of 4th Year Medical Students’ Competency in Prescribing
Authors: Soliman Youssef , Szarek John
Published: May 2022

PURPOSE

The Association of American Medical Colleges has delineated 13 Entrustable Professional Activities (EPAs) that entering residents should be expected to perform on day 1 of residency without direct supervision. EPA4, Enter and discuss orders and prescriptions, describes an activity that all recent medical graduates (Interns) spend a great deal of time daily on.

Additionally, with the ever‐increasing numbers of pharmaceuticals and biologics, there is a need for clinical pharmacology education in the curriculum to help students achieve EPA4. The Department of Medical Education at GCSOM partnered with the BPS to use their online platform in a two‐week clinical pharmacology elective for 4th year medical students. The hypothesis of this study was that completion of the program modules will enhance student learning and satisfaction with respect to prescribing.

METHODS

We used the BPS Assessment platform which provides prescribing skills modules for learners, which they access through a cloud‐based, content‐rich online assessment environment, with real‐time feedback. The assessments were reconciled to the US context insuring they were relevant to our students. Mean scores before and after completing the modules were compared using a paired t‐test. Students also completed a satisfaction questionnaire.

RESULTS

To date, 6 students have completed the program. The average scores (±SD) after completing the modules increased significantly (Before: 42%±14%; After: 74%±10%; p=0.001). This represented a normalized gain of 54%. Students tended to Agree/Strongly Agree to the 6 items related to the eLearning sessions, interface and assessment in the satisfaction questionnaire.

CONCLUSIONS

The results indicate that students’ knowledge and skills in prescribing increased as a result of their study of the modules. Their high ratings of the program suggest that students found it useful. In conclusion, the BPS program facilitates students’ abilities in prescribing therapies or interventions beneficial to patients and will be helpful in meeting the expectations of EPA4 as students enter residency.
Activation of the Central Medial Thalamic Afferent to the Orbitofrontal Cortex Contributes to Successful Reversal Learning
Authors: Tuite Kathleen , Girotti Milena , Morilak David
Published: May 2022

Stress‐related psychiatric disorders, such as major depressive disorder and anxiety disorders, have cognitive symptoms that are not well‐treated by classical antidepressants and cause major detriments to patients’ quality of life. Reversal learning, a form of cognitive flexibility necessary to adapt to a changing environment, is disrupted in stress‐related psychiatric disorders. The orbitofrontal cortex (OFC) mediates reversal learning, and hyperactivity in the OFC is associated with psychiatric disorders in humans. Using a reward‐based discrimination digging task to assess reversal learning in rodents, we have previously reported that chronic stress impairs reversal learning and that inducing long‐term depression in the mediodorsal thalamus to OFC pathway reverses these deficits. However, the circuit‐level mechanisms underlying reversal learning are not well established. Preliminary data using Fos immunohistochemistry, showed a significant decrease in Fos in the lateral OFC following reversal learning, and a significant increase in Fos induction in the central medial thalamus (CM). Converging evidence from other laboratories indicates the CM inhibits the prefrontal cortex. Therefore, we next tested the hypothesis that input to the OFC from the CM, inhibiting the OFC, contributes to successful reversal learning. We used an adeno‐associated virus to deliver an inhibitory (Gi) DREADD or GFP control construct into the CM under the control of the CaMKII promoter, and implanted guide cannulae into the lateral OFC for administration of the DREADD agonist directly at the DREADD‐expressing terminals from the CM. Animals received microinjections of DREADD agonist clozapine‐N‐oxide (300 µM, i.c.) directly preceding the reversal learning task. Activating the Gi DREADD significantly impaired reversal learning, increasing the number of trials it took animals to make six consecutive correct responses. These results suggest that excitatory input from the CM to the OFC is critical for successful reversal learning, indicating a novel role for the CM in cognitive flexibility. Future experiments will investigate the role of thalamic inputs to the OFC in the effects of stress on reversal learning.
Contribution Of Opioid Receptors To Antinociception Produced By ß‐Caryophyllene
Authors: Quintana Taylor , Sanchez Stephanie , Kandasamy Ram
Published: May 2022

Despite decades of research examining the biological mechanisms of pain, few new treatments have been developed. Cannabis has been used for centuries to treat pain, and previous evidence indicates that non‐∆9‐tetrahydrocannabinol and non‐cannabidiol constituents of Cannabis can produce pain relief in animal models. The Cannabis‐related terpene ß‐caryophyllene (BCP) produces pain relief in various pain tests with varying degrees of efficacy; however, the pharmacological mechanism underlying this pain relief is unclear. Previous studies by other groups have demonstrated that BCP produces antinociception via cannabinoid and opioid mechanisms following local administration into the hindpaw. As such, we hypothesized that BCP produces analgesia via opioid mechanisms following systemic administration. To test this hypothesis, we administered varying doses of BCP and evaluated their antinociceptive effects on the hot plate and tail‐flick tests in adult male Sprague‐Dawley rats. The hot plate test identifies pain relief mediated by supraspinal mechanisms. The tail‐flick test identifies pain relief mediated by spinal mechanisms. One intraperitoneal injection of 30 mg/kg BCP produced antinociception on the hot plate test and the tail‐flick test approximately 15 minutes after injection. Vehicle and doses of 3 mg/kg and 10 mg/kg were ineffective at producing pain relief on the hot plate and tail‐flick tests. In order to test the contribution of opioid receptors, a subset of rats received a 30 min pretreatment of the opioid receptor antagonist naltrexone. The effect of naltrexone on BCP‐induced antinociception varied depending on both dose and time. These results indicate that low doses of BCP may effectively alleviate acute pain and that BCP‐induced antinociception may be mediated, in part, by opioid receptors. Further studies are needed to fully understand the relationship between BCP and opioid receptors to determine their therapeutic potential.
Anti‐platelet effect of prednisolone is regulated by ERK‐mediated thromboxane generation
Authors: Kim Sanggu , Chaudhary Preeti K. , Kim Soochong
Published: May 2022

Endogenous hypercortisolism is associated with increased risk of blood‐related diseases such as high blood pressure and hyperglycemia that result in fatal cardiovascular disorders. Platelet plays a major role in hemostasis and thrombosis. Although it is well known that glucocorticoids exert a significant effect on hemostasis, the effect of endogenous cortisol on platelet function is much less clear.

We aimed to characterize the in vitro effect of prednisolone and its molecular basis on regulation of platelet function using murine platelets.

We first evaluated the concentration‐dependent effect of prednisolone on 2‐MeSADP‐induced platelet function and found that 2‐MeSADP‐induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2‐MeSADP‐induced secretion and the resultant secondary wave of aggregation is mediated by thromboxane A2 (TxA2) generation, this result suggested a role of prednisolone on TxA2generation in platelets. Consistently, prednisolone did not affect the 2‐MeSADP‐induced aggregation in aspirin‐treated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, AYPGKF‐ and thrombin‐induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive‐feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2‐MeSADP‐induced TxA2 generation confirming the role of prednisolone on TxA2 generation. Finally, Western blot analysis revealed that prednisolone inhibited the 2‐MeSADP‐induced ERK phosphorylation.

In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of ERK phosphorylation, thereby shedding light on the clinical characterization and its treatment efficacy in patients with cardiovascular disorders in the future.