bluuebunny/crossref_metadata_2025 huggingface dataset:
Beyond Place Cells: Using Algebraic Geometry to Determine Underlying Structure of Receptive Fields Associated with Sensory Neurons
Authors: Boucher Colleen J.
Published: May 2022

In 2014, John O’Keefe was jointly awarded a Nobel Prize for his 1971 discovery of place cells, a population of neurons in the hippocampus which contribute to an organism’s intuitive understanding of their position in relation to their surrounding space. Each place cell has a representative receptive field, the area in which the place cell fires when occupied by the organism, which based on their overlapping structure can provide us with information about an individual’s surroundings. The challenge of determining this overlapping structure, starting from neuronal firing patterns without context, still remains, but can be explored using algebraic geometry. Tools from algebraic geometry can describe the receptive fields of place cells to better understand the phenomena and how the signals of the brain contribute to the individual’s perception of their surroundings. To compute these relationships, we use the neural code, a binary representation of neurons firing; additionally, using Euler diagrams (a simple example being the Venn diagram) we can visualize the representation of the receptive field. Under certain constraints (i.e., connectivity), the overlap of receptive fields can be realized in up to three dimensions. This finding seems intuitive when applied to place cells since we perceive our position in respect to our surroundings in a three‐dimensional space. These same tools can be applied to other neuronal populations regardless of an intuitive dimensionality of the receptive fields. It is difficult to imagine a visual representation of all receptive fields, but with certain constraints we can construct a geometric visualization. To address this difficulty, we have developed a code using place cell data to compute the dimension of its receptive field. Then, using publicly available data sets, we used our code to determine the dimensionality of other sensory neuronal populations.
Inhibition of Ileal Bile Acid Transport is Protective Against Cholestatic Liver Injury in Cyp2c70‐/‐Mice with Humanized Bile Acid Composition
Authors: Truong Jennifer K. , Donepudi Ajay , Bennett Ashley , Malla Sudarshan , Zaufel Alex , Pachura Kimberly , Hellen Dominick , Moustafa Tarek , Dawson Paul , Karpen Saul
Published: May 2022

Cyp2c70 is the key murine liver enzyme responsible for the synthesis of 6‐hydroxylated muricholates. Cyp2c70 knockout (2c70‐/‐) mice present with a more human‐like bile acid (BA) composition devoid of protective, hydrophilic muricholic acids and with features of cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be protective in cholestatic models such as the Mdr2‐/‐ mouse. The aims of this study were to determine if IBAT inhibition is protective in 2c70‐/‐ mice and identify the underlying molecular mechanisms.

Methods

2c70‐/‐ mice (C57Bl/6J) were generated using CRISPR/Cas9 technology. Male and female wild type (WT) and 2c70‐/‐ mice were fed chow or chow containing 0.006% SC‐435 for 9 weeks following weaning at 3 weeks of age. Standard growth parameters were recorded; organs, gallbladder bile and blood were collected at necropsy for histologic, RNA and biochemical analyses.

Results

Male and female 2c70‐/‐ mice maintained on chow diet exhibited increased levels of serum liver injury markers ALT, AST, and BAs compared to WT mice. Evaluation of 2c70‐/‐ liver histology revealed increased hepatic inflammation, macrophage infiltration and bilary cell proliferation, which were markedly reduced with SC‐435 treatment. IBAT inhibition in 2c70‐/‐ mice improved liver histology, and reduced hepatic inflammation and bilary cell proliferation to levels indistinguishable from WT. The liver BA content was increased by 53% in 2c70‐/‐ mice, and was enriched in more hydrophobic BA species with an overall hydrophobicity similar that of humans (Figure 1). Treating 2c70‐/‐ mice with SC‐435 reduced the hepatic BA burden by 61%, but did not affect the overall hydrophobicity. RNA‐Seq and KEGG pathway analysis performed using liver RNA revealed genes involved in hepatic steroid and lipid metabolism (Sult2a1, Sult1e1, Hsd17b7), and detoxification (Cyp2c39, Cyp2c54, Cyp2c59, Cyp3a41a/b) were down‐regulated in 2c70‐/‐ mice and their expression were rescued with SC‐435 treatment. Conversely, genes in pathways related to immune cell activation and inflammation were significantly up‐regulated in 2c70‐/‐ mice and reduced to WT levels with IBAT inhibition (Figure 2).

Conclusions

Our findings indicate that reducing the hepatic BA burden without altering the hydrophobic compostion is sufficient to protect 2c70‐/‐ mice from hepatic injury. The findings suggest that there may be a threshold in liver for pathological accretion of hydrophobic BAs and reducing the hepatic BA pool is sufficient to alleviate liver injury, independent of hydrophobicity. Moreover, these data support the inhibition IBAT activity, and other approaches to reduce hepatic return of BAs in the enterohepatic circulation, as therapeutic targets to treat cholestasis.
The Metabolomic Pathways of the Senescence‐Associated Secretory Phenotype in C2C12 Myoblasts
Authors: Kamal Michael , Shanmuganathan Meera , Joanisse Sophie , Britz‐McKibbin Philip , Parise Gianni
Published: May 2022

Cellular senescence is considered a hallmark of aging that often occurs in tissues and cells in response to DNA damage. Most healthy cells become senescent after a fixed number of divisions or irreparable genomic damage, often because of external conditions such as circulating factors that are released by adjacent senescent cells. These secreted factors, known as the senescence‐associated secretory phenotype (SASP), are capable of in vivo reprogramming of otherwise healthy cells, ultimately inducing senescence. The involvement of cellular senescence in a variety of aging‐related diseases has been thoroughly examined, however the role of cellular senescence and the SASP in aging muscle remains understudied. Therefore, the objective of this study was to examine the role of metabolites as potential components of the SASP through an easily reproducible model of senescence in skeletal muscle myoblasts. C2C12 myoblasts were treated with an antitumour antibiotic, bleomycin, to cause DNA damage‐induced senescence, or with a vehicle control. Cells and associated media were collected 24 hours after treatment for metabolomic profiling using capillary‐electrophoresis – mass spectrometry (CE‐MS). Samples were compared to untreated myoblasts or fresh growth media and normalized to total cell count and protein content prior to analysis. Pathway analysis using the KEGG database revealed a substantial impact on amino acid metabolism within senescent cells, specifically phenylalanine and histidine metabolism. The media associated with senescent cells had a similar impact on amino acid metabolism, but also showed a significant impact on several polyunsaturated fatty acids, including arachidonic acid and docosahexaenoic acid. Notably, trimethylamine N‐oxide (TMAO), a metabolite previously shown to promote endothelial senescence/dysfunction and neuroinflammation, was detected at significantly higher levels in the media surrounding senescent myoblasts relative to vehicle‐treated controls (fold change = 2.878, p < 0.05). These data suggest that DNA damage‐induced senescence significantly impacts the metabolome of skeletal muscle cells. Future work will aim to evaluate the targeted impact of candidate metabolites as potential senescence inducers.
Small Brains, Small Skulls: Effect of Cortex Specific Overexpression of BAF170 on the Mouse Cranium
Authors: Coupe Abigail , Durham Emily L. , Lesciotto Kate M. , Veltri Megan F. , Kawasaki Mizuho , Motch‐Perrine Susan , Stoykova Anastassia , Tuoc Tran C. , Kawasaki Kazuhiko , Richtsmeier Joan T.
Published: May 2022

Cephalization is an important evolutionary and developmental process. Integration of the brain and skull throughout development and evolution produces brains that fit tightly within skulls. We sought to explore brain/skull integration using a mouse model that produces a small brain. Mammalian BAF170 is an intrinsic factor controlling cortical size, and cortex specific overexpression of BAF170 (conditional overexpression) which results in a mouse model in which cortical growth is inhibited. We looked at BAF170cOE mice and their unaffected littermates (controls) at various ages during cortical development, including embryonically at E15.5, as well as early post‐natal timepoints (P0, P14) as the cortex grows dramatically, and timepoints approaching complete cortical and skull growth (P28, P42). Using histological sections, we determined that the area of the cortex and number of nuclei within the cortex were reduced in BAF170cOE mice as compared to unaffected littermates, particularly at post‐natal time points. Using Euclidian Distance Matrix Analysis, we found no difference in skull form embryonically. Mediolateral dimensions of skulls of BAF170cOE mice were reduced by 3‐10% at P0 as compared to controls. At the later post‐natal time‐points (P14, P28, P42) there were significant differences in skull form between genotypes. Skulls of BAF170cOE mice were reduced by almost 20% along the anteroposterior and mediolateral axes. Our data show that the BAF170cOE mouse has a cortex that is reduced in size due to fewer nuclei resulting from overexpression of BAF170. The skull is not a direct target of the BAF170 gene, yet it responds to the change in brain size and shape by adjusting its size and contours to match the brain. The mechanisms that enable that coordination, a target for our future investigation, hold the key to understanding the evolution of human skull shape and encephalization. Additionally, these investigations may identify possible targets for treatment or prevention of craniofacial birth defects.
Engaging an Undergraduate Researcher to Develop a Mushroom Tyrosinase Assay as a Summative Assessment Device for an Upper‐level Biochemistry Laboratory Course
Authors: Hite Kristopher C.
Published: May 2022

Working with an undergraduate enrolled in 2‐credits of independent research a mushroom tyrosinase enzyme assay was researched, piloted, and optimized during the fall 2021 semester. The assay included crude mushroom extract from Agaricus bisporus, commonly known as white button mushrooms mixed with l‐3,4 dihydroxyphenylalanine (l‐DOPA) as a substrate. Change in absorbance was measured at 475 nm in a spectrophotometer. A version of this assay was then used to write and administer a summative assessment (a lab practical) in three sections of a 6‐credit capstone laboratory course for biochemistry majors. The practical evaluated students’ ability to measure enzyme reaction rates, make accurate dilutions, apply knowledge of Michaelis‐Menten kinetics to determine the Km and the Vmax of an enzyme of which they had little or no prior knowledge, and finally to gauge ability to determine the inhibition constant (Ki) of a provided inhibitor (kojic acid). While researching and optimizing this assay the undergraduate researcher gained hands‐on experience troubleshooting, preparing stock buffer solutions, realizing the limitations of certain substrate solubility, and observing the time constraints of an assay in which measurable products autocatalyze to form products that are not measurable by a spectrophotometer. The development of this assay and practical exam were both extremely student‐centered as the student gained practice judging the quality and feasibility of assays by searching and reading biochemistry and molecular biology education literature and communicating both via email and in‐person with corresponding authors. Generating new course content in a large lab courses with multiple sections is a challenge, especially in courses that have been taught for many years. Providing search parameters to an independent undergraduate researcher and suggesting journals to begin looking for feasible published protocols led to the implementation of an assay that piqued the interest of the student and lab instructors while also being useful as a tool for biochemistry laboratory assessment.
M‐Band Wavelet‐Based Imputation of scRNA‐seq Matrix and Multi‐view Clustering of Cells
Authors: Liu Zihuan , Liu Tong , Sun Wenke , Zhao Yongzhong , Wang Xiaodi
Published: May 2022

Wavelet analysis has been recognized as a cutting‐edge and promising tool in the fields of signal processing and data analysis. However, application of wavelet‐based method in single‐cell RNA sequencing (scRNA‐seq) data is little known. Here, we present M‐band wavelet‐based imputation of scRNA‐seq matrix and multi‐view clustering of cells. We applied combination of M‐band wavelet analysis and uniform manifold approximation and projection (UMAP) to a panel of single cell sequencing datasets by breaking up the data matrix into a trend (low frequency or low resolution) component and M‐1 fluctuation (high frequency or high resolution) components. Our hybrid analysis enables us to examine multi‐view clustering of cell types, identity, and functional states. Distinct to standard scRNA‐seq workflow, our wavelet‐based approach is a new addition to resolve the notorious chaotic sparsity of scRNA‐seq matrix and to uncover rare cell types with a fine‐resolution.
Sex‐dependent Regulation of Mitochondrial Respiratory Function in Mouse Brain Microvessels by Peroxynitrite Decomposition Catalyst
Authors: Sakamuri Siva S. , Sure Venkata N. , Kolli Lahari , Wisen William P. , Evans Wesley R. , Lindsey Sarah H. , Mostany Ricardo , Katakam Prasad V.
Published: May 2022

Background

Peroxynitrite (PN) is a strong oxidizing and nitrating molecule. PN is a potent inhibitor of mitochondrial respiration and promotes ischemia‐reperfusion injury following stroke. In isolated mouse brain mitochondria, we observed that PN donors inhibit mitochondrial respiratiory function whereas the PN decomposition catalyst, Fe (III) tetrakis (1‐methyl‐4‐pyridyl) porphyrin pentachlorideporphyrin pentachloride (FeTMPyP), enhances mitochondrial state III and state IVo mitochondrial respiration. In addition, we demonstrated that mitochondrial respiration is the primary contributor of cellular energy in brain microvessels (BMVs). The present study tested the hypothesis that FeTMPyP negatively regulates mitochondrial respiration in the mouse BMVs.

Methods

BMVs were isolated from male and female mice (C57Bl/6, 2‐4 months) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. BMVs were treated with FeTMPyP at 37oC for 60 minutes. Oxygen consumption rates (OCR) were measured using the Agilent Seahorse XFe24 analyzer and various respiratory parameters were determined following Mitostress test.

Results

In male BMVs, basal respiration, ATP production, and non‐mitochondrial respiration were not altered by FeTMPyP treatment. Contrary to our hypothesis, in male BMVs, PN decomposition catalyst decreased the mitochondrial maximal respiration by 24.6% (2.1 ± 0.4 vs 2.8 ± 0.3 picomoles of O2/min/µg protein;) whereas the spare respiratory capacity was reduced by 33.3% (1.2 ± 0.3 vs 1.8 ± 0.3 picomoles of O2/min/µg protein; n=17 each, p<0.05). Proton leak was elevated by 70% (0.7 ± 0.1 vs 0.4 ± 0.1 picomoles of O2/min/µg protein) by PN decomposition catalyst in male BMVs. In contrast, in the female BMVs, the PN decomposition catalyst failed to alter mitochondrial respiratory parameters (n=15 each, p=NS). Interestingly, FeTMPyP increased non‐mitochondrial respiration by 63.8% (0.95 ± 0.2 vs 0.58 ± 0.1 picomoles of O2/min/µg protein) in the female BMVs (n=17 each, p<0.05).

Conclusion

BMVs display sex‐dependent respenses to endogenous PN. Notably, in female mouse BMVs, PN appears to act as an antioxidant as PN inhibited the non‐mitochondrial respiration which mostly contributes to extramitochondrial superoxide generation.
Vitamin C Protects Against Doxorubicin‐Induced Muscle Oxidative Stress
Authors: do Nascimento Filho Antonio Viana , Stoyell‐Conti Filipe F. , Akolkar Gauri , de Miranda Victor Hugo M. , Singal Pawan , Irigoyen Maria Cláudia , De Angelis Kátia , da Silva Dias Danielle
Published: May 2022

Introduction

Doxorubicin, has been used as a chemotherapeutic agent, however, it is also associated with significant cardiotoxicity related to an increased oxidative stress (OS). Recent studies have shown that doxorubicin also induces skeletal muscle atrophy in preclinical models. Vitamin C (VC), which presents antioxidant properties, has been used as a pharmacological approach against cardiac toxicity. Nonetheless, the effects of vitamin C on doxorubicin‐induced skeletal muscle atrophy are still unknown.

Objective

Evaluate the effects of VC on OS parameters in skeletal muscle of rats exposed to doxorubicin.

Methods

Male Wistar rats divided into 4 groups (C: Control. VC: Vitamin C. D: Doxorubicin and VCD: VC+Dox, n=8‐10/group). Dox was administered in six doses of 2.5 mg/kg, for 3 weeks to obtain a cumulative dose of 15 mg/kg. VC was administered daily (50 mg/kg) orally for six weeks, starting one week before treatment with Dox. The gastrocnemius was weighed and homogenized for OS analysis: antioxidant profile (catalase [CAT], superoxide dismutase [SOD], glutathione peroxidase [GPx] and non‐enzymatic antioxidant capacity [FRAP]), pro‐oxidant (NADPH oxidase, hydrogen peroxide [H2O2], nitrites) and cell damage (protein and lipid oxidation [LPO]).

Results

Dox reduced the gastrocnemius weight, on the other hand VC prevented it (C: 1.74±0.03; VC: 1.55±0.18; D: 0.99±0.06 and VCD: 1.23±0.06 g). There were no differences in CAT, FRAP, NADPH and nitrites between studied groups. D group showed increased concentration of H2O2 compared to C and VC groups; which was not observed in the VCD group (C: 5.33±0.39; VC: 4.30±1.47; D: 8.72±1.58 and VCD: 3.66±0.73 μM H2O2). SOD was higher in the VCD group compared to the others (C: 8.29±0.61; VC: 8.38±0.48; D: 9.94±0.61 and VCD: 10.38±0.28 USOD/mg protein). GPx was higher in VC and VCD compared to C group and lower in D compared to C and VCD group (C:0.007±0.002; VC: 0.012±0.002; D: 0.009±0.001 and VCD: 0.011±0.001 nmol/mg protein). Protein oxidation was lower in C group compared to the others (C: 3.63± 0.93; VC: 6.98±0.74; D: 5.69±0.49 and VCD: 5.98±0.29 nmol/mg protein). However, LPO was higher in D group compared to C and VC groups, and lower in VCD group compared to D group (C: 342±122; VC:364±153; D: 998±131 and VCD: 389±50 cps/mg protein).

Conclusion

Vitamin C protects against doxorubicin‐induced skeletal muscle atrophy and oxidative stress, suggesting a potential approach to management cardio functional disorder in patients under doxorubicin treatment.
Lower blood pressure, but not muscle sympathetic nerve activity, in female compared to male adults during progressive central hypovolemia
Authors: Jarrard Caitlin P. , Trotter Claire E. , Crandall Craig G. , Watso Joseph C. , Huang Mu
Published: May 2022

INTRODUCTION

Rapid adjustments in cardiac output and sympathetically‐mediated vasoconstriction are critical to prevent blood pressure (BP) from falling during central hypovolemia. Female adults have a reduced ability to tolerate central hypovolemia (i.e., orthostatic hypotension), potentially due to reduced sympathetically‐mediated vasoconstriction. However, there are minimal data evaluating sex differences in muscle sympathetic nerve activity (MSNA) during pre‐syncopal limited central hypovolemia. Therefore, the purpose of this study was to compare autonomic cardiovascular responses during central hypovolemia between sexes. We tested the hypothesis that young healthy female, compared to male, adults would have a lower tolerance to progressive hypovolemia, accompanied by attenuated MSNA responses.

METHODS

Twenty‐eight female (28±8 years old, 170±7 cm, 75±8 kg, BMI: 26±3 kg/m2) and 31 male (age: 30±5 years old, height: 176±6 cm, mass: 82±9 kg, BMI: 26±3 kg/m2) adults completed a staged lower‐body negative pressure (LBNP) protocol. The LBNP protocol started at minus 40 mmHg (LBNP40) and was further reduced by 10 mmHg every 3 minutes until the participant reached pre‐syncope. MSNA (radial nerve), heart rate, and BP were collected continuously throughout the protocol. LBNP tolerance was quantified as a cumulative stress index (mmHg*min) and was compared between sexes using a two‐tailed Mann‐Whitney test. MSNA, heart rate, and BP responses during LBNP were compared between sexes (LBNP stage [repeated measure] x sex) using mixed‐model analysis of variance tests.

RESULTS

Consistent with prior findings, female adults had a lower tolerance to progressive LBNP (female: 513±283 vs. male: 929±357 mmHg*min, p<0.0001). Due to more females reaching pre‐syncope sooner in the protocol, we compared responses during the early stages of LBNP. At these early LBNP stages (LBNP40 & LBNP50), significant interactions for heart rate and BP were observed (heart rate: p<0.0001, BP: p=0.0464). Post‐hoc analyses revealed a significantly higher heart rate (98±20 vs. 82±15 bpm, p<0.0001) and lower mean BP (80±11 vs. 87±7 mmHg, p = 0.0343) in females at LBNP50. However, MSNA burst frequency responses were not different between sexes at the assessed time points (female vs. male; Baseline: 14±9 vs. 17±10, LBNP40: 29±14 vs. 34±12, LBNP50: 34±16 vs. 35±16 bursts/min; sex: p=0.2685, interaction: p=0.2334, stage: p<0.0001).

DISCUSSION

Consistent with previous reports, this study demonstrated a lower tolerance to progressive central hypovolemia in female compared to male adults. However, contrary to our hypothesis, there was no difference in MSNA burst frequency between sexes, despite lower BP in female adults during the early stages of LBNP. These findings support the need for future studies to explore other mechanisms contributing to sex differences in tolerance to central hypovolemia.
GPR30: A potential therapeutic target in Triple‐negative Inflammatory Breast Cancer
Authors: Bittman‐Soto Xavier S. , Rivera‐Lopez Adrian J. , Mendez‐Santacruz Laura A. , Peterson‐Peguero Esther A.
Published: May 2022

Inflammatory Breast Cancer (IBC) is rare and the most aggressive subtype of all breast cancers, with reported rapid progression, poor prognosis, and a unique clinical diagnosis (blockage of lymph vessels by tumor emboli). Approximately, 40% of IBC cases are classified as triple‐negative breast cancer (TNBC), meaning they test negative for hormone receptors (estrogen and progesterone) and the amplification of the epidermal growth factor receptor 2 (HER2), which result in no effective targeted therapies to date. However, 17β‐estrogen (E2) has been found to induce a non‐genomic cellular response to enhance oncogenic phenotypes in TN‐IBC and this has been associated with the presence of alternative estrogen receptors, including a novel G‐protein coupled estrogen receptor (GPR30). Recent studies have also reported a crosstalk between GPR30 and the epidermal growth factor receptor (EGFR) signaling pathway in the presence of E2, which enhances the phosphorylation of the extracellular signal‐regulated kinase (ERK1/2) and protein kinase B (AKT), and promotes migration and invasion phenotypes in TN‐BC cells. However, the exact role of GPR30 in the non‐genomic signaling pathway in TN‐IBC remains elusive. We hypothesize that GPR30 is necessary to undergo the estrogen non‐genomic signaling pathway to induce ERK1/2 and AKT phosphorilation levels and enhance migration and proliferation activities in TN‐IBC cells after treatment with E2.

Methods

We measured phosphorylation levels of ERK1/2 and AKT kinases (by western‐blot), migratory activity (by wound‐healing assay), and tumor emboli formation (by 3D‐cell proliferation assay) after treatments in time‐points with E2 alone and in combination with G15, GPR30 antagonist drug, in TN‐IBC cells SUM149PT.

Results

The combined G15 + E2 treatment significantly reduced E2‐induced ERK1/2 and AKT phosphorylation levels, as well as decreased migratory activity in SUM149PT cells, suggesting that GPR30 is necessary to undergo the estrogen non‐genomic signaling pathway and can regulate the ERK1/2 and AKT signaling cascade in TN‐IBC. Data acquired from these experiments will contribute to a better understanding of TN‐IBC’s molecular structure and create new opportunities to develop novel targeted therapies.
State‐dependent breathing emerges from interactions between repeated opioid use (ROU) and context
Authors: Szujewski Caroline , Wang Wesley , Otero Jose J. , Garcia Alfredo J.
Published: May 2022

ROU is arguably the single‐most influential factor driving respiratory‐related morbidities, overdose, and overdose‐related deaths in the ongoing opioid epidemic. When ROU is paired with the same environmental context (i.e., the drug‐paired context), it can produce pharmacodynamic tolerance to overdose‐related deaths. However, it is not clear if this context‐dependent tolerance impacts opioid induced respiratory depression (OIRD). This ongoing work examines how context pairing with ROU impacts breathing and OIRD. We developed a mouse model of ROU where five days of fentanyl administration (0.7 mg/kg) is paired with the same context. On subsequent testing days, OIRD in the fentanyl‐paired context (FP) was compared to OIRD in a novel environment. In the majority of mice (n=7 of 11), OIRD in the FP was smaller when compared to OIRD in the novel environment. Additionally, unsupervised machine learning of breathing metrics revealed that breathing patterns following ROU is distinct in FP as compared to that in the novel environment. These results indicate that behavior related to ROU impacts the consequence of opioids on breathing and produces predictable forms of state‐dependent breathing. Our findings may be leveraged to better predict and/or mitigate the occurrence of overdose‐related deaths associated with ROU.
Acute Antioxidant and Cytoprotective Effects of Caffeine in the Model of Neurovascular Injury by Oxidative Stress
Authors: Liu Jianxiong , Zhang Rong , Fedinec Alex L. , Parfenova Helena
Published: May 2022

Caffeine is commonly used in the neonatal intensive care units for respiratory disorders, but the effects of this medication in the neonatal cerebral circulation remain largely unexplored. We used newborn pigs as a clinically relevant model of the neonatal human brain to investigate the effects of caffeine on cerebral arterioles and primary neurovascular cells. First, we examined dose‐dependent cerebrovascular and systemic effects of water‐soluble anhydrous caffeine in anesthetized newborn pigs equipped with closed cranial windows. Caffeine administered systemically at therapeutic concentrations (20‐40 mg/kg i.v.) had no significant effects on diameter of pial arterioles, mean arterial blood pressure (MABP), and heart rate within 10‐60 min after the injection. When used at a higher concentration (70 mg/kg i.v.), caffeine produced dilation of pial arterioles (15‐20%) accompanied by an increased heart rate, without any effect on MABP. Topical application of caffeine (10‐5‐10‐3M) under the cranial window produced a dose‐dependent dilation of pial cerebral arterioles (15‐30%) that was fully reversed by a brief flushing with artificial cerebrospinal fluid. These in vivo data indicate that cerebral vascular cells are potential targets for caffeine. Next, we tested the hypothesis that caffeine exhibits cytoprotective properties in cerebral circulation during oxidative stress conditions. We used an in vitro model of acute oxidative stress‐induced neurovascular injury caused by excitotoxic and inflammatory conditions concomitant with epileptic seizures, asphyxia, and cerebral ischemia. Endothelial cells and cortical astrocytes are the key components of the neurovascular unit that are most vulnerable to oxidative stress injury. Primary cultures of cerebral vascular endothelial cells (CVEC) and cortical astrocytes from newborn pigs responded to pro‐inflammatory cytokine TNF‐alpha (30 ng/ml) and excitotoxic glutamate (1‐2 mM) by a surge in reactive oxygen species (ROS) production leading to apoptosis. NADPH oxidase is the major ROS‐generating system in the neurovascular unit during inflammation and excitotoxicity. We examined the acute effects of caffeine on oxidative stress‐induced neurovascular cell death caused by TNF‐alpha and excitotoxic glutamate. Caffeine (0.1‐10 µM) inhibited NADPH oxidase activation, reduced overall ROS generation, and attenuated major key events of apoptosis (caspase‐3 activation, DNA fragmentation, and loss of cell‐cell contacts) in CVEC and astrocytes exposed to TNF‐a and glutamate. These data demonstrate, for the first time, that caffeine exhibits acute antioxidant and cytoprotective effects in cerebral vascular endothelial cells and astrocytes. Overall, endothelial and astrocytic components of the neurovascular unit represent target cells for caffeine cytoprotective therapy in the neonatal cerebral circulation during oxidative stress.
Endothelial Pannexin1‐purinergic signaling pulmonary hypertension
Authors: Daneva Zdravka , Chen Yen‐Lin , Kuppusamy Maniselvan , Sonkusare Swapnil
Published: May 2022

Endothelial cell (EC) Ca2+ signals dilate small pulmonary arteries (PAs) and maintain a low resting pulmonary arterial pressure (PAP). Impairment of endothelial Ca2+ signaling mechanisms is a crucial contributor to elevated PAP in pulmonary hypertension (PH). Studies in systemic arteries show that flow/shear stress activates endothelial Ca2+ signals to cause vasodilation. Although PAs are a “high‐flow” vascular bed, flow‐induced endothelial Ca2+ signaling mechanisms have not been investigated in PH. In this regard, we recently showed that Ca2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4EC) ion channels dilates PAs and lowers resting PAP (Daneva et al., PNAS, 2021). Moreover, flow/shear stress activates the efflux of adenosine triphosphate (ATP) through endothelial Pannexin 1 (Panx1EC) in PAs. Panx1EC‐effluxed ATP, in turn, stimulates endothelial purinergic P2Y2 receptor (P2Y2REC) to increase TRPV4EC channels activity (Daneva et al., eLife, 2021). I hypothesized that impaired Panx1EC—P2Y2REC—TRPV4EC signaling axis reduces flow‐induced dilation of PAs and elevates PAP in PH. Confocal Ca2+ imaging studies in fluo‐4‐loaded en face PAs (~ 50 mm) showed that the baseline and agonist (GSK1016790A or GSK101)‐induced activity of TRPV4EC sparklets (individual Ca2+ influx signals through TRPV4EC channels) is reduced in the mice exposed to Sugen 5416 + chronic hypoxia (Su+CH; 3 weeks; 10% O2) compared to the normoxic control mice, indicating a decrease in TRPV4EC channel activity in PH. The expression of TRPV4 channels at the mRNA level, however, was not altered in Su+CH mice. The activation of TRPV4EC sparklets by exogenous ATP (1 mM) was also reduced in PAs from Su+CH mice, suggesting an impairment of ATP‐P2Y2REC‐TRPV4EC channel signaling axis in PH. Accordingly, ATP‐induced, endothelium‐dependent dilation was absent in PAs from Su+CH mice. We previously showed that basal ATP efflux in PAs predominantly occurs through endothelial Panx1, and flow/shear stress increases ATP efflux entirely through the activation of endothelial Panx1. Bioluminescence measurements of extracellular ATP concentration showed lower baseline ATP levels in PAs from Su+CH mice than control mice, suggesting a decrease in ATP efflux in PH. Importantly, flow/shear stress (4 – 15 dynes/cm2) increased the efflux of ATP from normal mice, but not in PAs from Su+CH mice, indicating that flow/shear stress‐Panx1EC signaling is also impaired in PH. Together, these data support the idea that flow‐Panx1EC and P2Y2REC‐TRPV4EC signaling are impaired in PH. Targeting individual elements of this pathway may provide novel therapeutic options in PH and other pulmonary vascular disorders characterized by endothelial dysfunction. Future studies will assess the individual signaling linkages of the flow‐Panx1EC‐P2Y2REC‐TRPV4EC signaling axis in PH.
15‐Days of Therapeutic Mild Intermittent Hypoxia Improves Cardiovascular, Upper Airway and Motor Limb Function in Participants with Motor Incomplete Spinal Cord Injury and Severe Obstructive Sleep Apnea
Authors: Panza Gino , Kissine Dylan , Puri Shipra , Lin Ho‐Sheng , Matieka Jason
Published: May 2022

Introduction

Repeated exposure to mild intermittent hypoxia (MIH) with sustained hypercapnia improves cardiovascular control in able‐bodied individuals with obstructive sleep apnea. Thus, we aimed to investigate if this treatment can improve cardiovascular function in individuals with motor incomplete spinal cord injuries (SCI). In addition, treatment with MIH improves cortical excitability, as well as, upper airway muscle and limb motor function. Consequently, we also explored if MIH improves upper airway motor function and walking endurance in individuals with motor incomplete SCI with concurrent sleep apnea.

Methods

Two individuals (age 51.0 ± 8.5) with traumatic motor incomplete SCI (T8, C6) and untreated severe obstructive sleep apnea (apnea hypopnea index 53.7 ± 34.0 events/hour) were treated with MIH for 15 days while receiving in‐home continuous positive airway pressure (CPAP) treatment. The MIH protocol consisted of twelve 2‐minute hypoxic exposures (PETO2 ≍ 50 mmHg) with PETCO2 maintained 2 mmHg above baseline. Therapeutic CPAP and the active critical closing pressure were measured before and after the 15‐day exposure period. In addition, blood pressure was measured over 24‐hours before and after the MIH protocol. Beat‐to‐beat blood pressure was measured during baseline (i.e. normoxia) prior to exposure to MIH on day 1 and day 15. Walking endurance was measured on the first and last day of the protocol prior to MIH exposure. Due to the small sample, Cohen’s D effect sizes are presented.

Results

24‐hour systolic (SBP), diastolic (DBP) and mean arterial (MAP) blood pressure (unit – mmHg) was reduced following 15 days of MIH (SBP: 131.4 ± 8.5 vs 113.4 ± 9.3, d= 2.0, DBP: 82.3 ± 1.4 vs 75.5 ± 3.9, d= 2.6, MAP: 98.5 ± 1.84 vs 88.0 ± 0.5, d= 9.04). Beat‐to‐beat blood pressure was also reduced following MIH (SBP: 135.3 ± 10.0 vs 116.9 ± 21.5, d= 1.2, DBP: 87.3 ± 0.46 vs 75.6 ± 3.1, d= 6.6, MAP 107.1 ± 0.4 vs 91.3 ± 7.0, d= 4.4). Additionally, an increase in indirect measures of cardiac output at rest (77.6 ± 39.9 vs 84.9 ± 30.2 L/min, d = 0.9) with concurrent reductions in total peripheral resistance (1.3 ± .7 vs 0.6 ± 0.1 mmHg*s/mL, d = 1.8) were found on the day 15. A reduction in therapeutic CPAP (11.0 ± 1.4 vs 9.0 ± 1.4 cmH2O, d= 1.4) and the critical closing pressure (4.5 ± 0.7 vs ‐0.6 ± 1.9 cmH2O, d = 3.9) was also evident following MIH. Lastly, walking endurance (155.8 ± 151.1 vs 240.5 ± 208.0 meters, d = 0.33) and walking velocity (0.4 ± 0.4 vs 0.7 ± 0.6 meters/second, d = 0.33) increased on day 15 compared to day 1.

Conclusion

Following 15 days of therapeutic MIH (i) blood pressure was reduced (ii) upper airway patency was improved and (iii) walking endurance and velocity increased in two individuals with motor incomplete SCI, hypertension and concurrent obstructive sleep apnea.
Trabecular and Cortical Bone Morphology of the Distal Radius in Humans, Chimpanzees, Cercopithecids, and Anteaters
Authors: Kuo Sharon , Saers Jaap P. , Scott Robert S. , Ryan Timothy M.
Published: May 2022

Identifying indicators of locomotor differences in extant and fossil taxa has been an ongoing endeavor in functional morphology. However, overlap between locomotion and phylogeny in primates makes overall interpretation of morphology, function, and behavior difficult. The distal radius is a particularly crucial area to understand loading patterns in the forelimb. This study aims to quantify trabecular and cortical bone properties in humans (bipeds), chimpanzees (knuckle‐walkers), cercopithecids (quadrupeds), and anteaters (quadrupeds) in order to discern different loading patterns in animals that vary in phylogeny and locomotion. Using a combination of whole bone analysis with point cloud correspondence registration and Bayesian mapping, we found locomotor signals in the trabecular and cortical bone properties. Importantly, we found a correspondence between cortical bone thickness and bone volume fraction in accordance with previously hypothesized patterns. Humans, unsurprisingly, have relatively low values for bone volume fraction and cortical bone thickness overall. Knuckle‐walking chimpanzees have increased cortical and trabecular bone along the palmar articular surface, reflecting increased loading in that region when compared to palmigrade or digitigrade cercopithecids. Anteaters have increased cortical bone thickness and trabecular bone volume fraction on the medial side of the radius, which reflects their unique loading patterns. Overall, trabecular and cortical bone variation in the distal radius appears to reflect differences in loading patterns due to differences in locomotion. Whole bone analysis of both cortical and trabecular bone can provide valuable insights into the skeletal response to locomotor loading and can be useful for reconstructing behavior in the past.
Arginine Vasopressin is not elevated in Early Pregnancy Loss
Authors: Santillan Mark K. , Shaw Kamara , Mulcahey Ashlyn , Leibold Emily , Coulter Kaylie , Funk Meghan L. , Hamilton Wendy , Brandt Debra , Santillan Donna A.
Published: May 2022

Background

Administration of arginine vasopressin (AVP) has been previously shown to influence myometrial contractility during the first trimester of pregnancy in women who were undergoing a planned therapeutic abortion. Consequently, it was hypothesized that AVP may play a role in early spontaneous pregnancy loss. AVP is made in a 1:1 molar ratio with copeptin; thus, copeptin can be used as a surrogate measure of AVP. While early pregnancy loss is likely a multifactorial problem, we sought to fill the gap in knowledge of the causes of using pre‐collected samples from pregnancies that subsequently underwent spontaneous abortion and matched controls.

Methods

A retrospective case control study was performed using plasma samples from the Maternal Fetal Tissue Bank (IRB# 200910784) at the University of Iowa Hospitals & Clinics. Whole blood samples were collected (ACD‐A tubes, Becton Dickinson) at confirmation of pregnancy appointments in which a viable pregnancy was detected by ultrasound (6‐10 weeks gestation). Samples were processed and plasma aliquots were snap frozen and stored at ‐80oC until analysis. Control samples (N=67) from viable, healthy pregnancies were matched by gestational age, race, ethnicity, and body mass index to cases (N=123). Copeptin was measured by an automated immunoassay (Brahms KRYPTOR) and total protein concentration via a bicinchoninic acid (BCA) protein assay (Pierce). Copeptin concentrations were normalized to total protein in samples. Continuous variables were analyzed by T test.

Results

No significant differences were detected in plasma samples that were collected while the pregnancies appeared viable. Copeptin was 7.2 +/‐ 6.0 mg/dL in case plasma samples and 6.0 +/‐ 3.4 mg/dL in control plasma samples (P=0.066). It is unclear if vasopressin increases acutely prior to the occurrence of spontaneous abortion.

Conclusion

These results suggest that vasopressin cannot be used to identify pregnancies at risk of early loss at the time of pregnancy confirmation.
The effect of brain serotonin deficiency on breathing during sleep is magnified by age
Authors: Pho Huy , Amorim Mateus R. , Qiu Qingchao , Shin Mi‐Kyung , Kim Lenise J. , Anokye‐Danso Frederick , Jun Jonathan J. , Ahima Rexford S. , Branco Luiz G. , Kuhn Donald M. , Mateika Jason H. , Polotsky Vsevolod Y.
Published: May 2022

Serotonin is an important mediator modulating behavior, metabolism, sleep, control of breathing, and upper airway function but the role of aging in serotonin‐mediated effects has not been previously defined. Our study aimed to examine the effect of brain serotonin deficiency on breathing during sleep and metabolism in younger and older mice.

We measured breathing during sleep, hypercapnic ventilatory response (HCVR), CO2 production (VCO2), and O2 consumption (VO2) in 16‐18 week old and 40‐44 week old mice with genetic deficiency of tryptophan hydroxylase 2 (Tph2), which regulates serotonin synthesis specifically in neurons, and Tph2+/+ controls.

In younger mice, Tph2knockout resulted in an increase in VCO2 and VO2 as a result of an increase in motor activity without any effect on HCVR and breathing during sleep. In contrast, in older mice, Tph2knockout caused not only increases in metabolic rate and motor activity but also weight loss, suppression of the HCVR, a decrease in maximal inspiratory flow during non‐rapid eye movement (NREM) sleep, and oxyhemoglobin desaturation during rapid eye movement (REM) sleep. Neither serotonin deficiency nor aging affected the frequency of flow limited breaths (a marker of upper airway closure) or apneas. Serotonin deficiency increased the amount and efficiency of sleep only in older animals.

In older mice, brain serotonin deficiency decreases hypercapnic sensitivity leading to hypoventilation and hypoxemia during REM sleep, which were absent at younger age. The serotonin pathway may play a role in the prevention of sleep‐disordered breathing in older individuals.
Enhanced Vascular Endothelial NLRP3 Inflammasome Activation by Plasma Exosomes from Liver‐specific Acid Ceramidase Gene Knockout Mice on the High Fat Diet
Authors: Yuan Xinxu , Wu Xiaoyuan , Li Pin‐Lan
Published: May 2022

Circulatory exosomes have been reported to induce vascular endothelial injury and neointima proliferation in mice with liver‐specific deletion of acid ceramidase gene (Asah1fl/fl/Albcre, Asah1 is mouse code of this gene). However, the precise mechanism mediating this exosome‐induced endothelial injury remains unknown. The present study tested a hypothesis that plasma exosomes from Asah1fl/fl/Albcre mice on the high fat diet (HFD) activate endothelial NLRP3 inflammasome activation leading to endothelial dysfunction. To test this hypothesis, wild type (WT/WT) mice and Asah1fl/fl/Albcre mice were fed with normal diet (ND) or HFD for 2 months. Using immunofluorescence confocal microscopy, we confirmed that ceramide levels significantly increased in the liver of Asah1fl/fl/Albcre mice compared to its WT/WT littermates and that HFD further enhanced lysosome ceramide levels in the liver as shown by the colocalization of ceramide with lysosome marker, lamp‐1. Then, isolated exosomes from the plasma of these mice were used to treat the primary cultured WT/WT endothelial cells from the carotid artery. It was found that exosomes isolated from HFD‐treated mice (HFD‐Exo) even from WT/WT mice significantly stimulated NLRP3 inflammasome formation and activation as shown by the significant increases in the colocalization of NLRP3 with Caspase‐1 or ASC, caspase‐1 activity and interleukin 1 beta (IL‐1β) level in the endothelial cells compared to exosomes from ND treated mice (ND‐Exo). This inflammasome formation and activation were remarkably enhanced if endothelial cells were treated by HFD‐Exo isolated from Asah1fl/fl/Albcre mice. To confirm whether lysosomal ceramide contributes to HFD‐Exo‐induced endothelial NLRP3 inflammasome activation, we further treated endothelial cells with C(2)‐ceramide, which was shown similar effects comparable to that induced by HFD‐Exo. Using dibutyryl‐cAMP, a blocker of ceramide‐induced action significantly attenuated C(2)‐ceramide‐ or HDF‐Exo‐induced enhancement of endothelial NLRP3 inflammmasome activation. Together, these results suggest that liver‐specific deletion of lysosomal Asah1 gene promotes release of HFD‐Exos that may contain high ceramide, activating endothelial NLRP3 inflammasome and thereby resulting in endothelial dysfunction during HFD‐induced liver steatosis.
Sex‐Dependent Effects of Gestational Intermittent Hypoxia Exposure in the 5XFAD Mouse Model of Alzheimer’s Disease
Authors: Ewald Andrea C. , Marino Kaitlyn M. , Romero Phinea Z. , McCann Erin C. , Baker Tracy L. , Ulland Tyler K. , Watters Jyoti J.
Published: May 2022

The in utero environment is well known to influence CNS development and behavioral phenotypes, lasting well into adulthood. However, long‐term neurodevelopmental reprogramming is poorly understood in the context of sleep‐disordered breathing (SDB) during pregnancy despite its increased prevalence in recent years. Neurodegenerative diseases, including Alzheimer’s disease (AD), strongly correlate with the incidence of sleep apnea in humans, a manifestation of SDB. To address this, we utilized a genetic mouse model of Alzheimer’s disease (AD; hemizygous 5xFAD) after in utero exposure to gestational intermittent hypoxia (GIH), a hallmark of SDB during pregnancy. We tested the hypothesis that GIH exposure will exacerbate the neurodegeneration previously described in 5XFAD mice. Pregnant dams received intermittent hypoxia (90s alternating 6.5%/21% O2) or normoxia (90s alternating 21%/21% O2) for 12 hrs/day during their sleep cycle from gestational days 10‐18. At 4, 6, and 8 months of age, offspring were subjected to prefrontal cortex‐ and hippocampal‐dependent behavioral testing to assess disease‐related decline. Preliminary data show GIH‐dependent decline in 5XFAD male offspring and not females. After final behavioral testing, brains were harvested for immunohistochemistry analysis and immunomagnetic microglial isolation. Imaging analysis demonstrated cellular alterations including increased number of microglia and decreased number of astrocytes in 5XFAD females exposed to GIH, which was not seen in males. Studies are underway to analyze microglial immune responses, inflammatory and intercellular communication gene expression by RNA‐sequencing. Together, these data suggest that GIH may exacerbate neurocognitive decline and alter cellular responses to disease that correlates with altered microglial function, in a manner that differs by sex. These observations have implications for people with sleep apnea, particularly during pregnancy and the subsequent brain health of their offspring.