bluuebunny/crossref_metadata_2025 huggingface dataset:
Immune Phenotypes of Oligodendroglial‐Lineage Cells in MDD and in Response to Chronic Stress‐Induced Microglial Inflammation
Authors: Madeira Miguel M. , Kokkosis Alexandros G. , Hage Zachary , Tsirka Stella E.
Published: May 2022

Background

Major Depressive Disorder (MDD) is a complex and heterogenous psychiatric disorder affecting more than 300 million people worldwide. Though the mechanisms underlying MDD are not fully understood, a subset of depressive patients can manifest chronic neuroinflammatory responses. In addition, brain‐imaging studies in MDD patients have provided evidence of white matter reductions and such changes are hypothesized to lead to disruption of Oligodendroglial‐Lineage cells (OLN) homeostatic mechanisms, resulting in destabilization of emotional/cognitive circuitry. The present goal of the study is to investigate whether these histopathological alterations are linked with chronic inflammation through single‐nucleus transcriptomics of MDD patients and in a depression mouse model.

Methods

Bioinformatics: Raw counts were downloaded from GEO (GSE144136), and expression objects created using Seurat. OLN were subsetted, clustered (using UMAP), and annotated based on their expression of early and late OLN markers. Pseudotime trajectory analysis was conducted using Moncle3. In vivo: The Repeated Social Defeat Stress (RSDS) paradigm (10 days) was used to induce depressive‐like behavior in 8–12‐week‐old male CX3CR1‐GFP+ and CSPG4‐EGFP+ mice. Behavioral tasks (BH) were performed to stratify the defeated mice to susceptible (S; depressive‐like) and resilient (R; non‐depressive) to stress groups. The study focuses on the MDD‐affected prefrontal cortex (mPFC) area (Fig).

Results

Separate clustering of MDD and Control samples yielded four similar clusters labeled based on their gene expression as: OPCs, Committed OPCs, Immature Oligodendrocytes (OLs), and Mature OLs. One MDD‐specific cluster was also obtained and labeled as Immune OLs (ImOL), given its expression of immune genes, such as: ARGHAP24, ADAM28, LPAR6, C3, CD74, and P2RY12. Further analysis of the ImOL cluster revealedexpression of markers associated with later phases of OLN progeny. Finally, a predictive model of pseudotime trajectory revealed alterations in the progression of OLN progeny in MDD samples. We then sought to identify this novel subset of ImOL in the RSDS model of depression. MHCII, C3 and P2RY12 were examined in conjunction with OLN markers (CSPG4, O4, CNP), with significant changes observed in S groups. Interestingly, microglial phagocytosis of myelin elements (CX3CR1‐GFP, MHCII, CNP), was significantly increased in S groups post RSDS, rendering the ImOL populations as potential recruiters of innate immunity.

Conclusions

Together, our computational data in MDD patients and depression mouse model, show that OLN can swiftly respond to chronic stress, and adopt an immune‐like phenotype, potentially contributing to the white matter disturbances observed in MDD.
Delivering MicroRNA‐137 to the Brain via Nanoparticles
Authors: Palumbo Michelle , Janowsky Aaron
Published: May 2022

Genetic mutations of microRNA‐137 (miR‐137) are strongly associated with symptoms of schizophrenia. Elevated miR‐137 impairs neuronal, dendritic, and synaptic development. Additionally, miR‐137 regulates multiple key mRNA transcripts that are critical for cognitive and sensory processing, and that are common genes dysregulated in schizophrenia. However, there remains a major barrier to treatment of schizophrenia due to the lack of targeted delivery of effective therapeutics to the brain. Encapsulating miR‐137 in nanoparticles offers a drug delivery system that can pass the blood‐brain‐barrier, allowing miR‐137 to have effects in the brain. We hypothesize that inhibiting endogenous levels of miR‐137, with the antisense sequence (anti‐miR‐137), may improve neurobiological and behavioral deficits as a result of miR‐137 overexpression. We encapsulated miR‐137 in nanoparticles and analyzed the size, polydispersity index, zeta potential, uptake efficiency, cargo delivery, and toxicity of miR‐137 loaded nanoparticles after treatment in cell culture. Results indicate nanoparticles are nontoxic delivery vehicles and are easily incorporated into cells. Additionally, the miR‐137 cargo is released from the nanoparticles and is detected as elevated miR‐137 levels. Following in vitro studies, we will test the most efficient delivery method to deliver nanoparticles to the brain in vivo by florescent labeling and ex vivo nanoparticle tracking. Delivering therapeutic RNAs to the brain will improve targeted drug delivery systems for central nervous system disorders.
An Accessory Muscle of the Pectoral Region
Authors: Moses Harrison , Ermler Melanie , Phan Brandon , Nazeer Cloe , Heukwa Anne , Barremkala Malli
Published: May 2022

Among the variations of pectoral muscles, this case is unique in the literature. An accessory pectoral muscle located deep to the left pectoralis minor, and attached to ribs 4‐6 near the midclavicular line, was discovered during a routine anatomy dissection. The muscle fibers do not fuse with fibers from other muscles, yet they run parallel to pectoralis minor. The accessory muscle can be distinguished from pectoralis minor because it has its own investing fascia and because subcutaneous fascia lies posterolaterally to the accessory muscle. These features place it in a distinct plane with unique innervation. This muscle holds clinical importance in that it may be a consideration for surgeons performing procedures including pectoral flap surgeries. Further, breast imaging may be complicated by such unexpected variations in the muscle tissue.
Deletion of the Transient Receptor Vanilloid‐1 (TRPV1) Channel in Rats Attenuates 2‐Kidney‐1‐Clip Hypertension
Authors: DeLalio Leon , Stocker Sean
Published: May 2022

Renal denervation lowers arterial blood pressure (ABP) in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves that increase sympathetic efferent activity and ABP. Renal sensory nerves highly express TRPV1 channels, and administration of the TRPV1 agonist capsaicin increases renal sensory nerve activity. However, the extent by which TRPV1 channels directly contribute to renal nerve dependent models of hypertension has not been tested. To test this hypothesis, we generated a novel Trpv1‐/‐ rat using CRISPR/Cas9 and deletion of exon 3. Male and female Trpv1‐/‐ and wild‐type littermates (8‐12 weeks) were instrumented with telemetry. At 2 weeks later, renovascular hypertension via renal stenosis was produced by placement of a PTFE cuff (0.16 x 0.22 inches, 1mm long) around the right renal artery. Male Trpv1‐/‐ and wild‐type rats had no differences in baseline mean ABP (99±2 vs 98±3 mmHg, respectively; n=7‐9) or heart rate (390±7 vs 400±8 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, mean ABP was significantly lower at Day 28 in male Trpv1‐/‐ versus wild‐type rats (125±8 vs 155±2 mmHg, respectively: P<0.01). Ganglionic blockade with chlorisondamine (2.5mg/kg, sc) at Day 28 produced a smaller fall in mean ABP of male Trpv1‐/‐ versus wild‐type rats (‐53±4 vs ‐86±3 mmHg, respectively; P<0.001). On the other hand, female Trpv1‐/‐ and wild‐type rats had no differences in baseline mean ABP (102±2 vs 104±1 mmHg, respectively; n=6‐9) or heart rate (419±8 vs 410±7 bpm, respectively). Renal stenosis significantly increased mean ABP in both groups; however, there were no differences at Day 28 between female Trpv1‐/‐ versus wild‐type rats (117±8 vs 122±6 mmHg, respectively). Moreover, the increase in mean ABP was smaller in females versus males. The ganglionic blocker chlorisondamine produced similar depressor responses in female Trpv1‐/‐ versus wild‐type rats (‐64±7 vs ‐65±7 mmHg, respectively). These findings illustrate a sex difference in renovascular hypertension in rats, but importantly indicate that Trpv1‐/‐ channels contribute to the established phase of renovascular hypertension in male rats.
Antagonism of Na,K‐ATPase signaling by pNaKtide can attenuate systemic inflammatory response in murine sepsis model induced by cecal ligation and puncture
Authors: Pereira Duane G. , Pillai Sneha S. , Lakhani Hari Vishal , Wang Jiayan , Sodhi Komal
Published: May 2022

Introduction

Oxidative stress and associated inflammatory processes are crucial in the development and progression of sepsis, resulting in an immune response that leads to multiple organ failure or dysfunction. Hence, the strategies to limit this systemic inflammatory response might result in the development of an effective sepsis therapy. Previous studies from our lab have demonstrated the role of Na/K‐ATPase signaling in exacerbating systemic oxidative stress and inflammation and the potential role of Na/K‐ATPase signaling antagonist, pNaKtide, in ameliorating several pathophysiological abnormalities.

Hypothesis

The aim of this study is to demonstrate the role of Na,K‐ATPase signaling in exacerbating oxidant stress and inflammatory response noted in a murine model of sepsis. Furthermore, we aim to demonstrate the effect of systemic administration of Na,K‐ATPase signaling antagonist, pNaKtide, in experimental sepsis using cecal ligation and puncture (CLP) model, as a drug intervention against septic shock.

Method

Murine sepsis was induced by CLP in male C57BL6 mice with or without pNaKtide (25 mg/kg body wt) which was administered, intraperitoneally, 24 hours before CLP procedure. Sham surgery was performed without the ligation and puncture, which served as controls for the study. All mice were assessed for Murine Sepsis Score (MSS) at baseline (before Sham or CLP), at 4hours, 8hours and 24 hours after Sham or CLP. Mice were euthanized after 24 hours of Sham or CLP surgery and tissues were collected for morphological and biochemical assessment. Blood was used to measure plasma levels of inflammatory cytokines.

Results

Systemic administration of pNaKtide demonstrated improved MSS at 24 hours following CLP surgery, as compared to CLP mice without pNaKtide. Histological assessment of lung tissues by H&E staining showed significantly less congestion, edema, infiltration of inflammatory cells and hemorrhage in CLP mice administered with pNaKtide, as compared to CLP alone. pNaKtide also improved systemic inflammatory cytokines assessed by plasma levels of TNFα and MCP1, as well as improved mRNA expression of inflammatory and macrophage infiltration markers, in lung tissues of CLP mice.

Conclusion

Our study demonstrates that antagonism of Na,K‐ATPase signaling by pNaKtide may attenuate CLP‐induced sepsis by inhibition of inflammatory milieu noted in this pathophysiological condition. Hence, Na,K‐ATPase signaling may serve as a viable clinical target for therapeutic intervention of sepsis and associated inflammatory mechanisms.
Mutagenesis and Enzyme Inhibition Studies of West Nile virus NS2B/NS3 Serine Protease
Authors: Calderon Kristen , Mitchell Jon C.
Published: May 2022

West Nile Virus (WNV) is the leading cause of mosquito‐borne disease in the United States, however there is currently no vaccine that can be used on humans to prevent or treat the infection. WNV is a +RNA virus of ~11 kbp and translated from a single open reading frame to produce a single polyprotein. This polyprotein is post‐translationally processed into three structural and seven non‐structural proteins. Specifically, NS2B and NS3 WNV proteins correspond to a serine protease which is required for further WNV viral replication (Nitsche 2018). Inhibition of this serine protease therefore represents an outstanding opportunity to possibly develop antiviral therapy. This research uses PCR to isolate a functional serine protease (NS2B/NS3), when expressed in the model bacterial organism, Escherichia coli (Shannon, 2016). An NS2B/NS2 serine protease assay will confirm the functionality of the enzyme. Utilization of known serpin (serpin protease inhibitors) will be used as positive control for the assay (Sanchez‐Navarro, AS et al., 2021). Utilization of the validated assay for NS2B/NS3 targeted mutagenesis through CRISPR/Cas9 or chemical mutagens, resulting in the identification of amino acid residues critical for functional WNV serine protease. These residues taken together with protein modelling and structure‐based design experiment, could then be used for drug development to generate novel chemical compounds or identify alternative natural product serpins (e.g. from bacteria, fungi, plants) which could ultimately prevent the virus from carrying out its final steps in replication and therefore inhibiting its ability to infect its host.
The impact of cyclooxygenase inhibition with ketorolac on regional cerebral perfusion in healthy adults
Authors: Muer Jessica D. , Didier Kaylin D. , Wannebo Brett M. , Carter Katrina J. , Hagen Scott A. , Eldridge Marlowe , Al‐Subu Awni M. , Livett Timothy A. , Wieben Oliver , Schrage William G.
Published: May 2022

Introduction

Non‐selective cyclooxygenase (COX) inhibition tests the role of COX on cerebrovascular control. Most studies using COX inhibitors (typically Indomethacin) demonstrate robust decreases in cerebral blood flow (CBF) (20‐42%), but have focused primarily on middle cerebral artery (MCA) velocity, which does not quantify CBF or provide insight into regional differences in the influence of COX on basal CBF control. The purpose of this study was to determine global and regional microvascular effects of COX inhibition to gain a better understanding of mechanisms underlying resting CBF control.

Hypothesis

We hypothesize that microvascular CBF will be attenuated after ketorolac infusion globally and in discrete regions.

Methods

Young healthy adults (M=4, F=2) were included in the study (age: 24 ± 3, BMI: 22.4 ± 0.7). Participants completed two Arterial Spin Labeling (ASL) scans. ASL scans were completed before and after a 3‐minute intravenous infusion of ketorolac (0.495 mg/kg, mean dose 33.9 ±2.8 mg). ASL analysis was completed in SPM12. ASL analysis included smoothing grey matter, co‐registering and normalizing raw data, brain masking and region of interest processing. A two‐way repeated measures ANOVA with a Tukey’s post hoc were computed in SigmaPlot13.

Results

Global cerebral blood flow during room air (58±15mL/100g/min) did not change after ketorolac infusion (57±24mL/100g/min)(p=0.83). Regional cerebral blood flow for pre‐ketorolac infusion and post‐ketorolac infusion were not significantly different((PRE mL/100g/min, POST mL/100g/min(p‐value)): Frontal lobes(Left:66±15, 65±23(p=0.87),Right: 68 ±15, 66 ±25(p=0.62)),Temporal lobes(Left: 56±12, 54±22(p=0.82); Right: 56±14, 54±24(p=0.61)),Parietal lobes (Left:56±12, 57±25(p=0.95); Right: 59±16, 59±26(p=0.99)), Occipital lobes(Left: 51±15, 54±30(p=0.58); Right: 50±16, 54±32(p=.44))).

Discussion

Our findings suggest ketorolac does not alter microvascular perfusion in healthy adults, suggesting not all COX inhibitors act on cerebral vasculature equally. These data add to previous work by comprehensively interrogating the entire brain rather than flow (or velocity) through single arteries.

Conclusion

There were no effect of ketorolac on global or regional cerebral blood flow in healthy adult
Endothelial KIR channel dysfunction in aged cerebral parenchymal arterioles
Authors: Behringer Erik J. , Hakim Md A. , Blackwell Jade , Pires Paulo W.
Published: May 2022

Aging is associated with cognitive decline via mechanisms that are not fully understood. Endothelial dysfunction is known to occur in parenchymal arterioles, the bottlenecks of the cerebral microcirculation. Dilation of these arterioles is highly dependent on changes in extracellular K+ ([K+]E), caused by activity of inwardly rectifying K+ channels (KIR2) or by opening of Ca2+‐activated small‐ and intermediate‐conductance K+ channels (KCa2.3 / KCa3.1, respectively). The effects of aging on activity of these channels in the endothelium of cerebral parenchymal arterioles remain undetermined, as well as their impact on endothelium‐dependent dilation. Thus, we hypothesized that advanced age will impair both KIR2 and KCa2.3 / KCa3.1 function in parenchymal arterioles of Aged mice (>24 months) when compared to Young (4‐6 months). Data are means ± SEM, Young vs. Aged males and females. No sex differences were observed, thus the data were combined. Using electrophysiology, we observed that freshly‐isolated endothelial cell tubes from Aged mice showed a similar hyperpolarization to the purinergic receptor agonist 2‐methyl‐S‐ATP (1 µM), which purportedly activates the downstream effectors KIR2 and KCa2.3 / KCa3.1, than those from Young mice (Δmembrane potential (Vm): ‐21.0 ± 1.7 vs ‐23.9 ± 5.1 mV, n = 5‐7), although vasodilatory responses of pressurized parenchymal arterioles were smaller (%Vasodilation: 22.4 ± 3.8 vs. 12.7 ± 3.2%, p<0.05, Student’s t‐test, n = 7‐6). Activity of KCa2.3 / KCa3.1 after activation by NS‐309 (1 µM) was unchanged by aging, as evidenced by similar hyperpolarization (ΔVm: ‐36.1 ± 2.4 vs. ‐32.4 ± 2.2 mV, n = 13‐14) and vasodilation (%Vasodilation: 16.5 ± 2.0 vs. 21.3 ± 3.2%, n = 8‐6). Activation of KIR2 by 15 mM [K+]E induced a smaller hyperpolarization in Aged than Young (ΔVm: ‐11.0 ± 1.1 vs ‐6.9 ± 0.7 mV, p<0.05, Student’s t‐test, n = 13‐13), with a paradoxical larger vasodilation that was unchanged by endothelium removal (%Vasodilation: 10.7 ± 2.3 vs. 25.0 ± 3.6% vs. 27.3 ± 4.5, one‐way ANOVA, Young endothelium intact vs. Aged endothelium intact vs Aged endothelium removed, n = 7‐13‐6), suggesting a compensation by smooth muscle KIR2. Lastly, spontaneous myogenic tone was higher in parenchymal arterioles from Aged mice, and endothelium removal did not further enhance myogenic tone (%myogenic tone: 25.4 ± 1.7 vs. 34.3 ± 2.3 vs 36.4 ± 3.2%, p<0.05, one‐way ANOVA, Young endothelium intact vs. Aged endothelium intact vs Aged endothelium removed, n = 22‐23‐6). In conclusion, aging impairs endothelial KIR2 channels and increases resting myogenic tone, which may underlie vascular dysfunction and exacerbate cognitive decline.
Epigenomic mechanisms used by KrasG12D to regulate inflammatory gene clusters in epithelial pancreatic cancer cells, which are critical for reprogramming the tumor microenvironment
Authors: Du Meijun , Abrudan Jenica L. , Urrutia Guillermo , de Assuncao Thiago Milech , Mathison Angela J. , Zimmermann Michael T. , Lomberk Gwen , Urrutia Raul
Published: May 2022

Kras is one of the most frequently mutated genes in pancreatic cancer, with the G12D variant acting as a cancer driver mutation. Our previous work has integrated and defined gene networks induced by KrasG12D expression, particularly the expression of inflammatory gene clusters. Moreover, we observed that KrasG12D expression increases the overall protein levels of several histone marks, including H3K4me3, H3K27ac, H3K27me3, H3K9me3 and integrated data analysis correlated gene activation and silencing with the alteration of histone marks. Here we further consider KrasG12D‐driven changes in gene expression and hypothesize that G9a‐H3K9me2 may play an important role in the epigenomic regulation of this process. To investigate epigenomic mechanisms underlying KrasG12D‐induced gene expression networks, CUT&RUN technology was used to profile H3K4me3 (promoter), H3K9ac (promoter, enhancer), H3K27ac (enhancer, super‐enhancer), and H3K9me2 (silencer) histone marks at regulatory elements. We used a genetically engineered mouse model (GEMM)‐derived inducible KrasG12D cell line and compared the epigenetic changes after 24hr of oncogene induction. Data was analyzed by using Sparse Enrichment Analysis for CUT&RUN (SEACR). At the genomic level, we found most H3K4me3 peaks (~90%) were close to a transcription start site (TSS), and the number of H3K4me3 peaks were reduced ~9x after KrasG12D activation. However, for the gene silencing mark, H3K9me2, >80% peaks were located at intronic or inter‐genetic regions, and the number of peaks were increased >5x with KrasG12D. For H3K9ac and H3K27ac histone marks, we observed more peaks at intronic or inter‐genetic regions than at the TSS, indicating the exchange of histone marks at enhancer, super‐enhancer and silenced regions may play an important role in KrasG12D‐induced gene expression changes. We next focused on histone modifications at regulatory elements of inflammatory gene clusters, such as IL11, CXCL9‐CXCL10‐CXCL11, and IL4‐IL13 gene clusters. We further correlated epigenetic modifications with our gene expression data and found that the H3K4me3, H3K9ac, and H3K27ac, and H3K9me2 histone mark pathways are involved in the regulation of these inflammatory genes. Notably, these pathways are both, critical for pancreatic cancer development and potentially druggable, and therefore, these results bear both mechanistic and biomedical relevance to pancreatic carcinogenesis.
Dissecting The Structural Contribution of The Cofilin N‐Terminus to Actin Filament Severing and Phosphorylation by LIMK
Authors: Sexton Joel , Turk Ben
Published: May 2022

Cofilin/ADF (actin depolymerizing factor) is a small actin‐binding protein important for cytoskeletal regulation within eukaryotic organisms. Cofilin promotes actin filament severing by disrupting interactions between adjacent actin subunits. Localized regulation of actin dynamics by cofilin phosphorylation is key to cell motility. Cofilin severing activity, and its regulation by phosphorylation, is mediated by an essential actin binding site within its N‐terminus (acetyl‐Ala2‐Ser3‐Gly4‐Val5 in humans). Phosphorylation of Ser3 by LIM domain kinases (LIMKs) is sufficient to inhibit actin binding and severing. Despite the importance of the cofilin N‐terminal peptide sequence, its functional contribution to the mechanism of actin severing has yet to be fully explored. We hypothesize that the cofilin N‐terminus contains evolutionarily conserved structural features that are necessary for actin severing activity and its regulation by LIMK. To identify key features of the cofilin N‐terminal sequence, we constructed a saturation mutagenesis library randomizing the first, third, and fourth residues of human cofilin, while maintaining a phosphorylatable Ser or Thr as the third residue. This combinatorial library of 16,000 (2 x 203) variants was screened as a pool for growth rescue in a strain of S. cerevisiaecontaining a silenced COF1gene. In parallel, we conducted the screen in yeast expressing exogenous human LIMK1 to identify cofilin variants that were inhibited by LIMK activity. In the absence of LIMK, the screen selected cofilin sequences with a Gly as the third residue and beta‐branched or hydrophobic residues for the fourth residue, including the native sequences from animal, plant, and yeast cofilins. In yeast expressing LIMK1, otherwise functional cofilin sequences containing a serine as the phosphoacceptor residue were unable to rescue growth. In contrast, Thr residues at the phosphoacceptor position and bulky hydrophobic residues at the third position imparted a protective effect that allowed cofilin variants to rescue growth despite the presence of LIMK activity. While LIMK1 was unable to phosphorylate a cofilin mutant with a Thr phosphoaccetor in vitro, large hydrophobic residues were tolerated at the third position, suggesting that they render cofilin functional even when phosphorylated. These results expand our understanding of actin regulation by providing structural insights about cofilin and by extension other proteins containing ADF‐homology domains.
Cholinergic‐mediated Circulatory Responses at the Onset of Isometric Exercise: Does Sex Matter?
Authors: Samora Milena , Teixeira André L. , Sabino‐Carvalho Jeann L. , Vianna Lauro C.
Published: May 2022

Introduction

We have shown that handgrip exercise evokes an initial decrease in systemic vascular resistance (SVR), which was abolished by cholinergic blockade (glycopyrrolate) and, thereby, augmented the blood pressure (BP) response. These findings suggest that a cholinergic mechanism is important for the BP and SVR responses at the onset of isometric handgrip exercise in humans. Noteworthy, this previous study included men only, and was not designed to investigate the potential existence of sex‐related differences in immediate BP and circulatory responses evoked at the exercise onset. Given this, the aim of this study was to test the hypothesis that women present an attenuated fall in SVR and increased pressor response compared to men at the onset of isometric handgrip exercise due to a blunted cholinergic vasodilation.

Methods

Ten healthy men (aged 21 ± 1 year) and 10 women (aged 24 ± 1 year) were recruited. Women were non‐oral contraceptives users and performed experiments in the early follicular phase of the menstrual cycle. Participants performed 20s bout of isometric handgrip exercise at 40% of maximal voluntary contraction. Beat‐to‐beat heart rate (HR) (ECG) and mean BP (finger photopletysmography) were continuously measured. Beat‐to‐beat stroke volume (SV) was obtained with the Modelflow method and matched with HR to estimate cardiac output (CO) and SVR. The exercise responses were expressed as a change from 10s immediately before the onset of voluntary contraction.

Results

The BP response pattern appeared to be different between men and women at the onset of isometric exercise. While men presented a typical exercise‐evoked biphasic BP response (3s: Δ2 ± 2 mmHg; 10s: Δ1 ± 1 mmHg; 20s: Δ5 ± 3 mmHg), women showed a progressive increase in mean BP (3s: Δ1 ± 1 mmHg; 10s; Δ3 ± 1 mmHg; 20s: Δ6 ± 1 mmHg; P=0.681). Men demonstrated a decrease in SVR at 3s (Δ‐0.9 ± 0.3 mmHg.L‐1.min), 10s (Δ ‐1.6 ± 0.4 mmHg.L‐1.min) and 20s (Δ‐1.9 ± 0.7 mmHg.L‐1.min) following the exercise onset, and these responses tended to be attenuated in women (3s: Δ‐0.3 ± 0.3 mmHg.L‐1.min; 10s: Δ‐1.2 ± 0.4 mmHg.L‐1.min; 20s: Δ‐0.6 ± 0.5 mmHg.L‐1.min; P=0.131). There were no sex differences in HR, SV and CO responses to exercise.

Conclusion

These findings suggest a lack of sex differences in immediate BP and circulatory responses at the onset of isometric handgrip exercise, indicative of similar cholinergic‐mediated vasodilation in skeletal muscle between men and women.
Hydrogen‐Deuterium Exchange Identifies the Structural Basis of Phase Separation of the Chromosome Passenger Complex
Authors: Bryan Nikaela W. , Niedzialkowska Ewa , Stukenberg P. T. , Black Ben E.
Published: May 2022

The centromere is the locus that directs accurate chromosome segregation, and a master regulatory complex, the chromosome passenger complex (CPC) is enriched in a region of the mitotic centromere termed the inner centromere. In mitosis, the CPC monitors the connections of the centromere to the mitotic spindle, acting to destabilize incorrect attachments but permitting correct attachments to persist. The CPC contains the Aurora‐B kinase and three regulatory subunits: INCENP, Survivin and Borealin. Recent findings support a role for CPC phase separation in its localization and function in mitotic error correction (Trivedi et al., 2019, Nat. Cell Biol. 21:1127‐1137). Liquid‐liquid phase separation is emerging as a central regulatory principle of cell biology. This form of phase separation is often driven by multivalency of adhesive domains and/or linear motifs. The sequences of these domains are often found in unstructured regions where they are enriched in uncharged polar side chains, charged amino acids, or aromatic residues; additionally, structural plasticity allows them to generate multiple, weakly‐adhesive, inter‐ and intramolecular interactions. Currently there are no techniques to globally map the motifs on proteins that drive the multivalent interactions. In this context, we now employ hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) to study dynamics of CPC phase separation. We have identified four regions within the regulatory CPC subunits that upon phase separation exhibit differential HX (three slower and one faster). Importantly, three of these regions map to structured regions of proteins while one maps to the expected intrinsically disordered regions. Each of the four regions is enriched with charged amino acids. Mutation of key residues in one of the differential HX regions within the INCENP subunit diminish droplet size and quantity, suggesting that we have successfully mapped a key surface for the phase separation properties of the CPC. In broader context we have shown for the first time how HXMS coupled with structural data and bioinformatic analysis can be applied to study molecular basics of protein liquid‐liquid phase separation.
Effects of a novel AMP‐mimicking prodrug on AMPK and mTORC1 activity in C2C12 cells
Authors: Ashby Jared S. , Pace Tyson W. , Allen Avery N. , Wilde Kody M. , Galvan David A. , Beitler Alyssa M. , Hancock Chad R. , Saunders Oliver , Thomson David M.
Published: May 2022

The beneficial effects of exercise on skeletal muscle health and function are well‐established. The intracellular signaling that mediates exercise’s effects on muscle are undoubtedly complex and are not entirely understood. Nonetheless, increasing adenosine monophosphate (AMP) levels during muscle contraction signals a declining energy charge in the cell and this certainly drives many of the beneficial adaptations of exercise. AMP‐activated protein kinase (AMPK) is one of AMP’s best‐defined targets and has been described as the cell’s master energy regulator. Because it is activated by exercise, AMPK has gained recent attention as an attractive pharmacological target in harnessing at least some of the benefits of exercise for those who cannot exercise due to old age or other disorders. The prodrug AICAR effectively activates AMPK as it is taken up into the cell and phosphorylated to form ZMP, an AMP mimetic. However, AICAR’s therapeutic potential is severely limited by its poor pharmacokinetics. Here, we describe a novel approach to ZMP delivery in a novel prodrug (Prodrug‐39; P39). We found that similar to AICAR, P39 concentrations above ~400 μM effectively achieved significant AMPK activation and glycogen depletion. Surprisingly, given AMPK’s well‐documented inhibition of the mechanistic target of rapamycin (mTORC1), P39 was also effective at stimulating p70S6k and rpS6 phosphorylation concentrations as low as 10 uM and early in the timecourse after administration of P39 at higher concentrations. Given this evidence, we conclude that Prodrug‐39 is indeed an effective AMPK activator and may also have some utility in mTOR activation at lower concentrations.
SPECC1L deficiency causes shortened primary cilia and genetically interacts with IFT‐A deficiency to affect palatogenesis
Authors: Hufft‐Martinez Brittany M. , Goering Jeremy P. , Stestiv Marta , Moedritzer Michael J. , Wang Henry H. , Tran Pamela V. , Saadi Irfan
Published: May 2022

Orofacial clefts are among the most common congenital malformations, affecting approximately 1 in 700 births. SPECC1L mutations in patients with syndromic cleft lip and/or palate cluster in the second coiled coil domain (CCD2), which facilitates interaction of SPECC1L with microtubules. We have shown that loss of SPECC1L in a null allele (Specc1l∆Ex4) resulted in perinatal lethality and delayed palatal shelf elevation, but no cleft palate (CP) or exencephaly at birth. However, mice with an in‐frame deletion of SPECC1L‐CCD2 exhibited CP and exencephaly, which are common manifestations of ciliopathies. Given that SPECC1L cytoplasmic expression includes localization around the cilia base and that SPECC1L regulates actin cytoskeleton, we investigated an association between SPECC1L and primary cilia. We observed that primary cilia were shortened relative to wild‐type in E13.5 Specc1l∆Ex4/∆Ex4 as well as Specc1l∆CCD2/∆CCD2 mutant palatal mesenchyme and epithelium. The cytoplasmic expression of SPECC1L around the cilia base was diminished in Specc1l∆CCD2/∆CCD2 cells. Thus, loss of SPECC1L association with microtubules appears sufficient to prevent its pericentriolar localization and to cause cilia shortening. Super‐resolution imaging for acetylated‐α‐tubulin and ciliary membrane protein, ARL13B, revealed bulbous structures in Specc1l mutant cilia, suggesting a defect in ciliary protein trafficking. Due to the overlapping phenotypes of shortened cilia, exencephaly and CP between the intraflagellar transport (IFT)‐A mouse mutant, Thm1aln/aln, and Specc1l∆CCD2/∆CCD2, we crossed these mutants to look for a genetic interaction. 18% of Specc1l∆CCD2/+, 0% of Thm1aln/+, and 33% of double heterozygous Specc1l∆CCD2/+;Thm1aln/+ embryos showed CP, confirming a genetic interaction. This was observed when the mother was Specc1l∆CCD2/+. However, remarkably, if the mother was Thm1aln/+, there was no CP incidence in Specc1l∆CCD2/+ and Specc1l∆CCD2/+;Thm1aln/+ embryos even though they had shortened cilia, suggesting Thm1aln/+ mothers provided a protective environment against CP phenotype. Consistent with our previous data, Specc1l∆Ex4;Thm1aln/+ mice resulted in no CP. These results suggest that shortened cilia inSpecc1l mutants do not cause but rather modulate CP phenotype. Overall, our studies indicate a novel role for SPECC1L in ciliogenesis, a novel genetic interaction between Specc1l andThm1, and a novel protective maternal genetic effect of Thm1 in palatogenesis.
BRD4‐MK2 signaling: target for Crohn’s Disease‐associated fibrosis
Authors: Chulkina Marina , McAninch Steven , Li Yi , Koltun Walter , Yochum Gregory , Tian Bing , Zhou Jia , Beswick Ellen J. , Brasier Allan R. , Pinchuk Irina V.
Published: May 2022

Introduction

Fibrosis is the major complication of Crohn’s disease (CD). No targeted therapies are currently available to revert CD‐associated fibrosis. While inflammatory mechanisms in CD have been extensively investigated, understanding the pathogenesis of fibrosis is relatively limited. Mesenchymal cells are the major effectors in profibrotic processes in CD. A dramatic increase in α‐SMA expressing subsets of mesenchymal cells, also known as myofibroblasts (MF), is among the main hallmarks of profibrotic changes in CD. Mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) is involved in the increase of α‐SMAexpression by MFs. Along with an increase in MK2 activity in CD we also observed an increase in the bromodomain‐containing Protein 4 (BRD4), an important epigenetic reader. BRD4 is critical for the expression of enhancer‐mediated profibrotic genes and, thus, is a potential novel regulator of MK2. However, the presence of mechanistic link between BRD4 and MK2 activity with respect to profibrotic activation of MFs in CD is unknown. Herein, we tested hypothesis that BRD4 mediated increase in MK2 activity is critical to the profibrotic reprogramming of MFs in CD and this pathway has potential for novel anti‐fibrotic therapy.

Methods

Human normal (N)‐ and CD‐derived MFs were analyzed using RNAseq, cytokine/chemokine multiplex array, FACS, and immunofluorescent microscopy. The T cell transfer murine colitis model relevant to CD was also used in this study.

Results

An Increase in BRD4 and MK2 expression was observed in primary human CD‐ when compared to N‐MFs and were associated with the expression of the profibrotic genes including Col1α2, α‐SMA, TNC, Fn1, and TGF‐β1. Inhibition of MK2 activity in CD‐MFs with the MK2 specific inhibitor PF‐3644022 and murine MK2 KO MFs showed significantly reduced basal and TGF‐β1‐induced profibrotic gene expression. Use of the BRD4 specific inhibitor ZL0454 significantly reduced MK2 mRNA expression in CD‐MFs Additionally, ex vivo treatment of the CD tissues with ZL0454 also abrogated MK2 expression. Therapeutic use of the BRD4 inhibitor ZL0454 (10 mg/kg, daily for 10 days) in T cell transfer murine colitis significantly decreased MK2 expression and downstream profibrotic responses as measured by collagen deposition and expression of fibrosis‐related genes.

Conclusions

Our data suggests that BRD4 positively regulates MK2 expression in CD. Further, BRD4‐MK2 signaling is critical to profibrotic activation of mesenchymal cells in CD and is a potential therapeutic target to reverse CD‐associated fibrosis.
Superior Pharmacokinetic Profile of an Innovative, Next Generation Curcumin Formula
Authors: Rai Deshanie , Morde Abhijeet , Padigaru Muralidhara , Acharya Manutosh
Published: May 2022

Introduction

Curcumin is the major bioactive component of turmeric or Curcuma longa L., a widely used spice, food colorant, preservative as well as medicinal agent in Indian and Chinese remedies. Numerous studies have demonstrated its potential benefits for heart health, immunity, diabetes, arthritis as well as joint health and mobility. Currently, curcumin‐based products in capsules, ointments, tablets, and cosmetics are marketed worldwide. One important drawback of using curcumin is its poor bioavailability and hence the need to use it at high doses to achieve measurable plasma levels and efficacy – this limits both consumer compliance and product formulation. Curcumin’s poor solubility, low absorption from the gut, rapid metabolism and rapid systemic elimination contributes to its poor bioavailability. To help overcome this major limitation of curcumin, we developed a proprietary curcumin formulation (Curcuwin Ultra+) (CU+) to enhance its bioavailability. We hypothesized that the formulation would protect the curcuminoids from degradation at intestinal alkaline pH conditions, and through this mechanism, significantly increase the availability of curcuminoids in blood and at the tissue level.

Goal

Our goal was to measure the pharmacokinetic profile of CU+ vs. a standard turmeric extract (STE).

Method

A randomized, double‐blind, crossover study design involving 24 healthy volunteers under fasting conditions was implemented. Subjects received a single dose of 250 mg CU+, 500 mg CU+ and 1900 mg of STE. Blood samples before dosing, and at various time points post dosing up to 24 hours were collected. Total curcuminoids were measured using a validated LC‐MS/MS method. Formulations were compared based on Cmax, AUC0‐6, AUC0‐24, Tmax, t1/2, and relative absorption.

Results

Maximum serum concentration (Cmax) and total systemic exposure (AUC0‐6 and AUC0‐24) for total curcuminoids were significantly higher (p<0.05) for both doses of CU+ vs. STE. For example, AUC0‐6 was 144 and 149 times greater for CU+ at 250 mg and 500 mg respectively compared to 1900 mg of STE. Additionally, CU+ showed 40% faster absorption as measured by Tmax vs. the STE (p<0.05). No adverse events were observed.

Conclusions

These pharmacokinetic data demonstrates the ability of this curcumin formulation to deliver superior bioavailability vs. STE, and, at doses that are considerably lower than STE and several other commercially available curcumin products. This has important relevance to consumer compliance in terms of lowering curcumin dose and frequency of dosing. Since maximizing bioavailability influences the therapeutic effects of a compound, the next phase of our research program is to determine if this enhanced bioavailability can be translated to an efficacious benefit of CU+.
Deletion of RGS2 Results in Increased Blood Pressure and Depression‐Like Behavior in the Presence of Elevated Ang II Levels in Female Mice
Authors: Luu Anh , Waldschmidt Ryan , Guisinger Trevor , Seeley Sarah , D'Souza Manoranjan , Chrissobolis Sophocles
Published: May 2022

Hypertension and depression are major health problems in the US. Overactivity of the renin angiotensin system (RAS) plays an important role in the pathophysiology of hypertension and depression. The actions of the main effector peptide of the RAS, angiotensin II (Ang II), are mediated by the Ang II type 1 receptor whose activity is modulated by regulator of G‐protein signaling (RGS) proteins, including RGS2. In this study, we assessed the effects of genetic deletion of RGS2 on Ang II‐induced increases in blood pressure (using tail cuff plethysmography) and depression‐like behavior (using the tail suspension test) in adult male and female mice. Adult male and female RGS2 wild‐type (RGS2+/+) and RGS2‐deficient (RGS2‐/‐) mice were treated with osmotic minipumps containing either vehicle (saline) or Ang II (1 mg/kg/d) for 21 days. In males, baseline SBP was lower in RGS2‐/‐ (SBP = 103±5 mmHg, n=14) vs RGS2+/+ (SBP = 120±6 mmHg, n=13, P<0.05 vs RGS2‐/‐) mice. By contrast, in females there was no difference in baseline SBP between RGS2+/+ and RGS2‐/‐ mice, suggesting that deletion of RGS2 results in lower baseline SBP in females only. Chronic treatment with Ang II increased SBP in both RGS2+/+ and RGS2‐/‐ mice compared to saline controls, irrespective of sex. There was no difference in Ang II‐induced increases in SBP in RGS2+/+ and RGS2‐/‐ male mice, suggesting that RGS2 does not protect against Ang II‐induced increases in SBP in male mice. By contrast, in female mice, Ang II‐induced increases in SBP were greater in RGS2‐/‐ compared to RGS2+/+ mice (for example, after 21 days of Ang II treatment, change in SBP from baseline in RGS2+/+ mice was 47±11 mmHg, n=5, and in RGS2‐/‐ mice it was 73±15 mmHg n=4, P<0.05) suggesting that RGS2 protects against Ang II‐induced increases in SBP in female, but not male mice. Chronic Ang II treatment also resulted in decreased immobilization time in RGS2+/+ female mice, but not RGS2‐/‐ mice compared to respective saline controls (immobilization time in vehicle‐treated RGS2+/+= 187±14 sec, n=5; Ang II‐treated RGS2+/+= 124±27 sec, n=5, P<0.05 vs vehicle‐treated RGS2+/+; vehicle‐treated RGS2‐/‐= 196±16 sec, n=5; Ang II‐treated RGS2‐/‐ = 192±13 sec, n=4, P<0.05 vs Ang II‐treated RGS2+/+). These data suggest that RGS2 protects against depression‐like behavior in the presence of elevated Ang II levels in female mice. In contrast to female mice, Ang II treatment and RGS2 deficiency had no effect on depression‐like behavior in adult male mice. Overall, these data suggest that RGS2 protects against blood pressure increases and depression‐like behavior after exposure to elevated Ang II levels in female mice, suggesting that RGS2 activation is a promising therapeutic target to treat elevated blood pressure and depression during conditions of elevated Ang II levels in females.
Determining the Requirements for Gαi:β‐arrestin Complex Formation at G Protein‐Coupled Receptors
Authors: Kohlmann Taylor , Lee Claudia , Rajagopal Sudarshan
Published: May 2022

G protein‐coupled receptors (GPCRs) comprise the largest class of transmembrane receptors and are targeted by nearly a third of prescription drugs. Canonical GPCR signaling involves pathways mediated by G proteins and β‐arrestins (βarrs). While these have historically been thought of as separate, our lab has demonstrated that the inhibitory G protein Gα subunit (Gαi) and βarr form complexes following receptor activation. We have also shown that Gαi:βarr complex formation occurs even if the receptor signals through other classes of G proteins or lacks G protein activity altogether, as in the case of endogenously βarr‐biased Atypical Chemokine Receptor 3 (ACKR3). To expand on this work, we used a split nano‐luciferase assay to examine Gαi:βarr complex formation at βarr‐biased mutants of two ‘balanced’ receptors, which exhibit both G protein and βarr signaling— the type 1 Angiotensin II Receptor (AT1R) and D2 dopamine Receptor (D2R). Consistent with our results with ACKR3, we found that mutation of these other receptors to abrogate G protein‐mediated signaling did not eliminate Gαi:βarr association. Observing this at multiple additional receptors further supported the idea that G protein activity is not required for Gαi:βarr complex formation. To add to our characterization of the determinants of Gαi:βarr association, we then sought to explore how different mechanisms of βarr activation may contribute to complex formation at ACKR3. To accomplish this, we utilized previously described polar core, proximal finger loop, and lipid mutant βarr constructs. The polar core and proximal finger loop mutants achieve constitutive activation by different mechanisms, while the lipid mutant’s ability to be recruited to and bind at the plasma membrane is deficient; these result in decreased Gαi:βarr association at Vasopressin Receptor 2 (V2R). We compared these mutants’ ability to associate with Gαi at ACKR3. Our constitutively active βarr constructs showed differential abilities to form Gαi:βarr complexes compared to wild‐type βarr, suggesting that certain methods of βarr activation may be more relevant in Gαi:βarr complex formation than others. Additional insights into the determinants of Gαi:βarr association in the context of receptor, G protein, and βarr activity will enhance our understanding of the Gαi:βarr complex and its importance in GPCR signaling, potentially informing future development of therapeutic agents targeting GPCRs.
Modeling Alcohol and Amphetamine Co‐Use in Adolescent Spontaneously Hypertensive Rats
Authors: Telatin Marta , Rhoads Dennis E.
Published: May 2022

Non‐medical use of amphetamine and other stimulants prescribed for treatment of attention deficit/hyperactivity disorder (ADHD) peaks in adolescence and is of growing concern when combined with binge consumption of alcohol. Previous studies in our lab modeled chronic ethanol‐amphetamine co‐use in adolescent Long‐Evans (LE) rats and provided evidence that amphetamine attenuates alcohol withdrawal symptoms in a manner that could lessen an individual’s awareness of impending alcohol dependence. The objective of the current project is to determine if adolescents of the Spontaneous Hypertensive Rat (SHR), used as a model for ADHD, respond differently to ethanol‐amphetamine co‐administration. Adolescent Wistar‐Kyoto (WKY) rats were included as a control. Adolescent SHR and WKY rats at P32 were administered one of four liquid diets: control (no drug), ethanol (3.6%, w/v), amphetamine (20 mg/L), or ethanol combined with amphetamine. Rats were withdrawn from their respective diet at four different time points: 5, 12, 19, and 26 days, and tested for alcohol withdrawal symptoms. Computer‐controlled activity chambers equipped with a dark box insert were used to assess general locomotor activity and anxiety‐like behavior. Overall alcohol withdrawal severity was also evaluated. After 5 and 12 days of consuming alcohol, SHR adolescents showed marked hypoactivity typical of alcohol withdrawal but no anxiety‐like behavior. Hypoactivity declined with additional periods of ethanol administration and there was surprisingly low overall withdrawal severity after 26 days. Amphetamine co‐administration did not affect withdrawal hypoactivity or overall withdrawal severity. Anxiety‐like behavior became significant for SHR at the 19‐day time point but only in the groups with amphetamine plus ethanol and amphetamine alone, not when withdrawing from alcohol alone. This contrasted sharply with WKY adolescents which showed very high overall withdrawal severity at the longer time points and both hypoactivity and anxiety‐like behavior during withdrawal at the earlier time points. Of particular interest, each of these withdrawal behaviors was eliminated when amphetamine was co‐administered with ethanol. Thus, compared to WKY or LE, SHR adolescents appeared resistant to the progressive signs of alcohol withdrawal used to gauge alcohol dependency in rodents and showed unique anxiety‐like behavior when withdrawing from amphetamine with or without alcohol co‐administration. Current work is focusing on understanding the underlying biochemistry of these withdrawal behaviors in SHR adolescents and particularly the absence of alcohol withdrawal symptoms with its potential relevance for individuals with ADHD.