bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain Serotonin Deficiency and Fluoxetine Lead to Sex‐Specific Effects on Binge Eating in Mice
Authors: Pellechia Sophia A. , Baugher Brittany J. , Karth Melinda D. , Huq Shama , Warner Allison , Karth Michelle M. , Sachs Benjamin D.
Published: May 2022

Binge eating disorder (BED), the most common eating disorder in the USA, negatively impacts psychological and physical health. Although the neurobiological basis of BED remains unknown, serotonergic dysfunction has been hypothesized to contribute to this condition. This study tested the hypothesis that brain serotonin deficiency will increase binge eating and block reductions in binge eating induced by fluoxetine. Binge eating was measured in wild‐type and serotonin‐deficient mice after acute and chronic fluoxetine administration. RNA was isolated from the raphe, and real‐time PCR was performed to evaluate gene expression. Males with low serotonin binge ate more than wild‐type mice at baseline, and fluoxetine reduced binge eating in both genotypes. Gene expression analyses revealed no significant genotype or drug effects on p11 expression in males, but fluoxetine significantly reduced the expression of 5HT1A receptor but not 5HT2A. In females, fluoxetine reduced binge eating in wild‐type but not serotonin‐deficient mice, and no baseline difference in binge eating was observed. Real‐time PCR revealed no significant main effects of genotype or drug on p11, 5HT1A, or 5HT2A expression. However, a significant genotype by drug interaction was observed for p11 expression in which fluoxetine reduced p11 expression in wild‐type mice and increased it in serotonin‐deficient animals. These results highlight the importance of sex as a modifying factor in the relationship between binge eating and serotonin levels and suggest that serotonin deficiency can modify sensitivity to fluoxetine‐induced reductions in binge eating, perhaps by impacting transcriptional responses to fluoxetine.
EB3 inhibitor increases chromatin accessibility and reprograms neovascular endothelial cells
Authors: Chan Wan Ching , Lee Quinn , Le Jonathan , Maienschein‐Cline Mark , Komarova Yulia
Published: May 2022

In our previous studies, we described a critical role of microtubule‐associated end binding protein 3 (EB3) in regulating pathological calcium release from endoplasmic reticulum (ER) stores through interaction with inositol 1,4,5‐trisphosphate (IP3R3) within endothelial cells. The short cognate peptide we designed based on this interaction blocked VEGF‐evoked ER calcium release and decreased laser‐induced choroidal neovascularization (CNV) in both mouse and non‐human primate models of age‐related macular degeneration. Here we analyzed the global effects of this peptide on choroidal and retinal cells using single nuclei‐RNA (snRNA) and single nuclei‐assay for transposase‐accessible chromatin (snATAC) sequencing of non‐human primate eye tissue. Within the snRNA‐seq transcriptome data, we detected 18 different cell types residing in the retina and choroid using well‐established markers. For the characterization of snATAC‐seq clusters, we performed Pearson correlation of the genes expressed in each cluster of our snRNA‐seq data and the peaks found in each cluster of our snATAC‐seq data. Our snATAC‐seq analysis demonstrated a widespread increase in chromatin accessibility at the promoter region across different cell types. These changes in chromatin accessibility were associated with increased acetylation in lysine 27 of histone 3 (H3K27ac) but not lysine 8 of histone 4 (H4K8ac). Concurrent analysis between our snATAC‐seq and snRNA‐seq data further revealed increased chromatin opening of MEIS2 and PAX6 within our metabolic‐active endothelial cells of neovascularized lesions, leading to activation of downstream transcription factors such as NCOR2, NR2C1, SREBF2, TCF4, TCF12, TEF, and THRA. The activation of this transcriptional program promoted neovascular regeneration and healing of ablated regions within CNV lesion of non‐human primate eye in the treatment group. Taken together, we concluded that EB3 inhibition prevented aberrant angiogenesis through epigenetic reprogramming of injured endothelial cells and through activation of MEIS2 and PAX6’s transcriptional program.
Investigating the Determinants of Substrate Binding Orientation within the E. coli Hsp70 Chaperone Protein
Authors: Olds Nina , Clerico Eugenia M. , Gierasch Lila M.
Published: May 2022

Molecular chaperone proteins play a critical role in maintenance of the cell’s proteome through protein quality control. Heat shock protein 70 chaperones (Hsp70s) are an evolutionarily conserved and diverse family of chaperone proteins that assist with proper protein folding and prevention of aggregation in the cell. Previous research has shown that the substrate‐binding domain of the E. coli Hsp70, DnaK, contains binding pockets of low stringency extending bilaterally from a highly hydrophobic central pocket. The SBD interacts with short hydrophobic sequences of substrates by “reading” the sequence. The SBD will then bind the substrate in one of two orientations: an N‐C, or forward, orientation, or a C‐N, reverse, orientation. Research done thus far on substrate binding has not revealed an exact mechanism that determines orientation. In this study, peptides possessing the same sequence with opposing backbone directions were used to test the significance of backbone interactions with the SBD as well as residue side chains and their interactions with the SBD. The analysis of the bound orientation of these peptides is accomplished by disulfide cross‐linking using peptides containing an N‐terminal Cys and SBD proteins with a Cys introduced in such a way where it can be reached in only one orientation. The results indicated slight energetic favorable biases for the interactions of residue side chains and the SBD pockets. The results also place a high importance on leucine residues in substrates as a determinant of optimal pocket ‐ residue fit and binding orientation. Altogether, the data suggest that binding occurs in the orientation that supports the most optimal fit of residue side chains into binding pockets, which reveals further intricacies and selectivity involved in substrate binding for Hsp70s.
Mechanical Tension‐induced Vascular and Alveolar Morphogenesis during Lung Regeneration
Authors: Mammoto Akiko , Hunyenyiwa Tendai , Kyi Priscilla , Hendee Kathryn , Matus Kienna , Mammoto Tadanori
Published: May 2022

Mechanical forces altered after unilateral pneumonectomy (PNX) control post‐PNX vascular and alveolar morphogenesis. This study aims to understand the mechanosensitive mechanism of lung vascular and alveolar regeneration in the post‐PNX mouse lungs. Vascular formation is stimulated at the subpleural region in the remaining mouse lung after unilateral PNX, where mechanical stretch is enhanced, while inhibition of mechanical tension by insertion of prosthesis attenuates these effects. The focal adhesion protein, paxillin senses mechanical stretch after PNX and the levels of paxillin are upregulated in SPB‐positive alveolar epithelial type II (ATII) cells and endothelial cells (ECs), which stimulates the expression of angiogenic factor angiopoietin‐1 (Ang1) in ATII cells and Ang2 in ECs to induce EC directional motility to the subpleural region. Cellular stretching increases the expression of paxillin, which induces expression of Ang2 in ECs and Ang1 in ATII cells in vitro. Post‐PNX lung growth and vascular formation toward the subpleural region are suppressed in Pxnfl/fl‐Sftpc‐CreERT2 mouse lungs, in which paxillin expression is attenuated in ATII cells. Structure of lamellar bodies that control surfactant homeostasis is also disrupted in post‐PNX Pxnfl/fl‐Sftpc‐CreERT2 mouse lung ATII cells. These results suggest that cooperative action of mechanosensitive paxillin signaling in ECs and ATII cells mediates vascular and alveolar epithelial formation during post‐PNX lung growth.
The novel function of CUL4B via switch glucose metabolism to contribute to cancer progression
Authors: Li Chien Hsiu , Chan Ming‐Hsien , Hsiao Micheal
Published: May 2022

Metabolism reprogramming and excessive glycolysis are associated with rapid cancer cell growth, motility, and resistance to drug treatment. Therefore, identifying candidates for regulating the ratio of glycolysis and gluconeogenesis is a priority. Especially the reverse effect of Aldolase/Fructose‐1,6‐Bisphosphatase. Fructose‐1, 6‐bisphosphate (F‐1,6‐BP), is a vital intermediate involvement in glucose metabolism. When F‐1,6‐BP is converted into the 3‐carbon product by Aldolase, the glycolysis will proceed sequentially, while FBP1 can reverse the equation. Based on established immunoprecipitation and proteomic analyses revealed several candidates, including the anterior gradient 2 (AGR2), ubiquitin ligase gene cullin 4B (CUL4B), and telomeric repeat binding factor 1 (TERF1) that significantly modulates the ratio between ALDOA and FBP1. Our study demonstrated the inversed correlation of ALDOA and FBP1 associated with lung adenocarcinoma and hepatocellular carcinoma progression. Molecular simulated analysis and clinical correlation indicated that CUL4B is the novel factor in switching ALDOA and FBP1. Moreover, we evaluate the CUL4B‐relative glucose metabolism events to describe how CUL4B participates in the switch of ALDOA and FBP1. Based on these relationships, we claimed that CUL4B could trigger glucose metabolism reprogramming and be used as a therapeutic target for further research.
Single Cell Assessment of Mitochondrial Function
Authors: Mahadev Bhat Sanjana , Yap Jane Q. , Ramirez Ramirez Oscar A. , Delmotte Philippe , Sieck Gary C.
Published: May 2022

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.
Smooth Muscle TRPV4 Channels Signaling Nanodomains in Blood Pressure Regulation
Authors: Chen Yen‐Lin , Daneva Zdravka , Kuppusamy Maniselvan , Ottolini Matteo , Klimentova Eliska , Sokolowski Jennifer D. , Park Min S. , Sonkusare Swapnil K.
Published: May 2022

Transient receptor potential vanilloid 4 (TRPV4) channel is a crucial Ca2+influx pathway in arterial smooth muscle cells (SMCs). However, the effect of SMC TRPV4 (TRPV4SMC) channel activity on resting blood pressure has not been investigated. We hypothesized that SMC TRPV4 (TRPV4SMC) channels regulate blood pressure under homeostatic and hypertensive conditions. Radiotelemetric recordings showed lower resting blood pressure in SMC‐specific TRPV4‐/‐ (TRPV4SMC‐/‐) mice than control mice, suggesting that TRPV4SMC channels elevate the resting blood pressure. α1 adrenergic receptor (α1AR) stimulation–induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4SMC‐/‐ mice, whereas intraluminal pressure–induced (myogenic) constriction was increased. Moreover, both α1AR stimulation and increased intraluminal pressure activated elementary Ca2+ influx signals through TRPV4SMC channels (TRPV4SMC sparklets) in MAs from control mice. These data supported the concept that TRPV4SMC channels contribute to α1AR–induced vasoconstriction but oppose myogenic vasoconstriction. Inhibition of Ca2+‐sensitive K+ (BK) channels abolished the effect of TRPV4SMC channels on myogenic vasoconstriction, indicating a crucial role for TRPV4SMC–BK channel signaling in limiting myogenic vasoconstriction. Accordingly, a direct activation of TRPV4SMC channels with GSK1016790A (30 nM) increased BK channel currents in SMC patch‐clamp experiments. α1AR stimulation, however, was unable to increase BK channel currents, implying two functionally opposite pools of TRPV4SMC channels. Direct STORM superresolution imaging revealed a1AR:TRPV4SMC and TRPV4SMC:BK channel co‐localization, and almost no co‐localization of a1ARs and BK channels. Together, these data supported the idea of spatially separated and functionally opposite nanodomains of TRPV4SMC channels. We further hypothesized that an imbalance between these two nanodomains contributes to elevated blood pressure in hypertension. In skeletal muscle arteries from hypertensive patients, a1AR activation of TRPV4SMC channel currents was accentuated whereas TRPV4SMC activation of BK channel currents was impaired. Similarly, MAs from angiotensin II‐infused hypertensive mice showed increased activity of constrictor a1AR–TRPV4SMC signaling and impairment of dilatory TRPV4SMC–BK channel signaling. Collectively, our data identify novel smooth muscle Ca2+ signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.
Identification of Adipose‐Specific iNKT Cell Subpopulation through Single‐Cell RNA Sequencing
Authors: Han Sang Mun , Park Jeu , Park Eun Seo , Choi Yoon Ha , Huh Jin Young , Kim Jong Kyoung , Kim Jae Bum
Published: May 2022

Invariant natural killer T (iNKT) cells are innate T cells that recognize CD1d‐loaded lipid antigens. In adipose tissue, iNKT cells contribute to maintenance of adipose tissue homeostasis through active communication with CD1d‐positive adipocytes. By recognizing lipid antigens, adipose iNKT cells kill harmful cells, such as hypertrophic adipocytes and pro‐inflammatory macrophages, and promote stem cell proliferation. These characteristics seem not to be observed in hepatic or splenic iNKT cells. However, it has not been thoroughly understood how adipose iNKT cells control cell fates in adipose tissue. Here, we adopted single‐cell RNA sequencing to identify unique subpopulations of adipose iNKT cells involved in the clearance of deleterious cells or stem cell renewal. Adipose iNKT cells were largely categorized into 3 subpopulations; iNKT1A, iNKT1B, and iNKT17. Transcriptome analysis revealed that iNKT1A cells were the adipose‐specific subpopulation, while iNKT1B and iNKT17 cells were similar to splenic and thymic iNKT cells. Moreover, adoptive transfer experiments showed that iNKT1A cells were generated only in adipose tissue, but not in other organs, indicating that adipose tissue microenvironment plays crucial roles for generation of iNKT1A cells. Collectively, our data showed that iNKT1A cells would be the adipose‐specific iNKT subpopulation and could be generated by adipose tissue microenvironment.
Acute Low Dose Cannabidiol Treatment Boosts Social Interaction Preference in Male Mice
Authors: Gould Georgianna G. , Ferreira Livia F. , Pathapati Nikhita , Nunn Mary C. , Greene Susan M. , Ginsburg Brett C. , Schultz Stephen T.
Published: May 2022

Cannabidiol (CBD) is a non‐addictive psychoactive phytochemical from Cannabis sativa that is increasingly used to manage pain. The potential for CBD to ameliorate some psychiatric disorder symptoms has been suggested, including the social behavior deficits of autism. To test this hypothesis, adult male BTBRT+Itpr3tf/J (BTBR) mice, a model of idiopathic autism, were acutely treated with vehicle or 0.1, 1, or 10 mg/kg CBD. Social interaction preference was assessed 50 min after treatment, followed by social novelty preference at 60 min, marble burying at 75 min and social dominance at 120 min. CBD (10 mg/kg) enhanced BTBR social interaction but not social novelty preference, marble burying or dominance, with serum levels = 29 ± 11 ng/mg at 3 h post‐injection. Next, acute 10 mg/kg CBD was compared to vehicle treatment in serotonin transporter (SERT) knockout mice, as SERT deficiency is an autism risk factor, and in C57BL6/J mice. CBD treatment generally enhanced social interaction preference and attenuated social novelty preference, yet neither marble burying nor dominance was affected. These findings show acute treatment with purified CBD can enhance social interaction preference at a relatively low dose. However, the pharmacological target(s) by which this low dose of CBD enhanced sociability remain to be demonstrated. Any use of unregulated CBD products to treat autism symptoms in children is not advisable until product purity and dose are strictly controlled, and acute versus long term treatment effects are better known.
The Effects of Exercise Training with Oral Intake of Lactate on Hippocampal Neurogenesis and Neurotrophic Factors in Mice
Authors: Hwang Deunsol , Kyun Sunghwan , Jang Inkwon , Park Hun‐Young , Kim Jisu , Lim Kiwon
Published: May 2022

Hippocampal neurogenesis plays an important role in learning and memory. Healthy hippocampal environment is also necessary for improvement of its cognitive function. Therefore, enhancing of both hippocampal neurogenesis and environment is required to prevent or ameliorate neurodegenerative statements.

Exercise is powerful enhancer of both, and it is considered that the exercise effects are resulted from alterations in brain (central mechanism), also in soma, i.e., skeletal muscle, (somatic mechanism). Yet the exact mechanism is unclear.

Lactate, known as just byproduct of glycolysis and primary factor of fatigue when exercise, is highlighting as a promising signaling molecule that mediates the exercise effects on hippocampus. In this perspective, exercise training with intake of lactate can be remarkable strategy for strengthening the exercise effects on hippocampus.

Therefore, the purpose of the current study is to investigate whether combination of exercise and lactate intake can additively enhance exercise‐induced hippocampal neurogenesis and neurotrophic factors.

The study was conducted for 5 weeks using 4 groups of mice: CON (vehicle group), LAC (oral administration of 3g/kg lactate 5days/week), EXE (mid‐intensity exercise 5days/week), and EXE+LAC (lactate administration immediately after every exercise). At the last week of experiment, cognitive function was evaluated by Y‐maze spontaneous alternation test. After the experiment ended, hippocampal neurogenesis (Ki67, neural stem cell proliferation marker; DCX, marker of differentiation to immature neurons) and neurotrophic factors (PGC1α, FNDC5, VEGFA, and BDNF) were evaluated respectively by immunohistochemistry (IHC) and by Western blot (WB) on hippocampus.

From the IHC results, exercise training significantly increased the number of Ki67 and DCX positive cells (p= 0.001 and p = 0.004 respectively) which were also increased in LAC than CON (p = 0.064 and p = 0.03 respectively). There was no difference in Ki67 and DCX positive cells between EXE and EXE+LAC. From the WB results, however, FNDC5 of EXE+LAC was significantly higher than that of EXE (p = 0.005). BDNF and PGC1α of EXE+LAC was higher than that of EXE respectively by 20% and 9% (but not significant). There was no difference in VEGFA between EXE and EXE+LAC. From the Y‐maze test results, interestingly, while there was no difference in the performance between CON and LAC, the performance of EXE+LAC was significantly enhanced than that of EXE (p = 0.006).

In conclusion, combination of exercise training and lactate intake additively promoted the performance of spontaneous alternation test and tended to additively enhance exercise‐induced neurotrophic factors, but not hippocampal neurogenesis. Therefore, the study revealed the potential that exercise training with oral intake of lactate can be remarkable strategy for strengthening the exercise effects on hippocampus.
Chemokine Receptor Antagonism Decreases Drug Choice and Modifies Opioid Analgesia in Rats
Authors: Mason Briana M. , Collins Gregory T.
Published: May 2022

Substance use disorders (SUDs) and overdose deaths have reached unprecedented levels despite considerable efforts to develop pharmacotherapies for their treatment and prevention. Chemoattractant cytokines (‘chemokines’) are immune system messengers that can alter the therapeutic and abuse‐related effects of opioids and stimulants. Therefore, the aims of this study were to evaluate the effectiveness of Maraviroc, a CCR5 antagonist, and AMD3100, a CXCR4 antagonist, to alter 1) the self‐administration of fentanyl, an opioid, and cocaine, a stimulant, using a food versus drug choice procedure and 2) the analgesic effects of fentanyl in a radiant heat assay. Adult male Sprague Dawley rats were trained to respond under a fixed ratio (FR) 5 on one lever to receive an infusion of fentanyl (n=8), and on an alternate lever to receive food (grain‐based pellet). The choice procedure consisted of 5 sequential 20‐min components, each separated by a 2‐min intercomponent interval. Food was available across all five components, with increasing unit doses of fentanyl (0.00032‐0.01 mg/kg/infusion) available during components 2‐5; no drug was available during component 1. Once responding stabilized, rats underwent a series of drug pretreatment tests, including: naloxone (1, 3.2 mg/kg; IP), haloperidol (0.01‐0.1 mg/kg; IP), Maraviroc (1‐17.8 mg/kg; IP), and AMD3100 (1‐17.8 mg/kg; IP). A separate cohort of 8 adult male Sprague Dawley rats was used to evaluate the effects of Maraviroc (10 mg/kg; IP), and AMD3100 (10 mg/kg; IP), on paw withdrawal latencies in a thermal nociception procedure following cumulative doses of fentanyl (0.01‐0.1 mg/kg; IP). In rats responding for food or fentanyl, naloxone reduced fentanyl choice while increasing choice of food, whereas haloperidol had little effect on the choice for fentanyl, but decreased responding on both levers at large doses. Maraviroc and AMD3100 had similar effects all rats tested, effectively reallocating behavior away from the drug and towards the food reinforcer. At the largest dose tested, Maraviroc (17.8 mg/kg; IP) disrupted behavior in the early portions of the session, suggestive of a sedative effect in some animals. Maraviroc and AMD3100 did not significantly modify the analgesic effects of fentanyl during tests of thermal nociception. These data suggest that antagonism of CXCR4 and CCR5 may be of use in the ongoing effort to develop medications for the treatment of SUDs.
Role of apoptosis and autophagy in masseter muscle atrophy evoked by Botulinum Toxin injection
Authors: Quezada Esteban R. , Chiong Mario , Buvinic Sonja
Published: May 2022

Introduction

Musculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy.

Aim

To evaluate the induction of apoptosis and autophagy in BoNTA‐injected masseter muscle of adult mice.

Methodology

Unilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC‐Universidad de Chile, #21446‐ODO‐UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2‐7d post‐injection and immunofluorescence at 7d post‐injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t‐test, one‐way ANOVA test, Mann‐Whitney test, or Kruskal‐Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4‐8; p <0.05).

Results

Unilateral injection of BoNTA did not change the relative levels of apoptosis‐related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86‐fold), non‐lipidated LC3 (1.66‐fold), and P62 (1.24‐fold) at 7 d post‐injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction.

Conclusions

The atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy‐ or apoptosis‐induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.
Lactate Administration Increases Skeletal Muscle Synthesis in Mice
Authors: Kyun Sunghwan , Jang Inkwon , Hwang Deunsol , Park Hun‐Young , Kim Jisu , Lim Kiwon
Published: May 2022

Purpose

Lactate, secreted by skeletal muscle during exercise, had been known as fatigue substance. However, several physiological roles of lactate have been revealed in the last decades, and previous studies reported that lactate treatment increased protein synthesis factors in skeletal muscle. Nevertheless, underlying molecular mechanisms of exogenous lactate on protein synthesis and degradation remain unknown, and there was no study that confirm long‐term lactate administration with exercise training. Therefore, the purpose of study is to investigate the effect of long‐term exogenous lactate administration and lactate administration with exercise training on the skeletal muscle synthesis and degradation.

Methods

32 mice were randomly divided into four groups: sedentary + saline (Sed/Con), sedentary + lactate (Sed/Lac), exercise training + saline (Ex/Con), and Exercise training + lactate (Ex/Lac). Exercise training (VO2max 70%) and lactate administration (3 g/kg sodium lactate) performed 5 days a week. The timing of lactate administration was immediately after every exercise training, and saline administration groups were given same volume of saline as lactate. After 5 weeks, mice were sacrificed and immediately obtained skeletal muscle and blood. To confirm the effect of lactate on protein synthesis (IGF receptor, Akt and mTOR phosphate/total) and degradation (MuRF1, MAFbx1, FOXO1), mRNA and protein expression analyzed respectively plantaris and extensor digitorum longus. Blood parameters of lactate, insulin, IGF1, and glucose were measured.

Results

Skeletal muscle weight tended to higher Sed/Lac than Sed/Con, but Sed/Lac had significantly supplement effects compared to Sed/Con in only gastrocnemius weight (p = .000). Blood IGF1 showed that Sed/Lac was significantly lower than Sed/Con (p = .039) and Ex/Lac (p = .007). mRNA of IGF receptor had only exercise effects (p = .000). mRNA of Akt and mTOR showed that Sed/Lac was significantly higher than Sed/Con (respectively, p = .000 and p = .043). Protein expression of MuRF1, Sed/Lac was significantly lower than Sed/Con (p = .046), but exercise training effects was found saline groups (Sed/Con vs Ex/Con; p = .001).

Conclusion

This study showed that long‐term lactate administration had positive effects on Akt / mTOR and MuRF1. Especially, it was confirmed that long‐term lactate administration increased skeletal muscle mass in mice. Hence, we suggest that long‐term lactate administration increases skeletal muscle synthesis and that lactate administration can be used as supplement for sarcopenia. Nevertheless, this study did not confirm synergistic effects of lactate administration with exercise training. It is suggested that post‐exercise lactate administration, unlike lactate administration at rest state, affects energy metabolism rather skeletal muscle synthesis. Therefore, future study would be necessary to investigate lactate administration timing with exercise training.
Methionine‐aromatic Interaction as a Tool to Enhance Protein Stability
Authors: Chatterjee Kiran Sankar , Das Ranabir
Published: May 2022

Nonbonded aromatic interactions play a significant role in stabilizing a folded protein structure(1, 2). Apart from well‐studied aromatic‐aromatic interactions in proteins, preferential proximity of sulfur containing cysteine and methionine sidechains and aromatic rings is also observed in protein hydrophobic cores and at the interfaces of protein‐protein complexes. Methionine‐aromatic motifs are examples of such S‐aromatic motifs that provide additional stabilization at a longer distance range over pure hydrophobic interactions(3). Although the idea of preferential association of aromatic amino acids has been successfully implemented for de novo design of peptides and proteins, the additional stability provided by met‐aromatic motif over pure hydrophobic interaction and its potential significance in designing a stabler protein fold has not been examined before. The present work shows the relevance of S‐aromatic interaction in increasing thermodynamic stability of a protein fold using Small Ubiquitin‐Like Modifier (SUMO) protein. We found that a highly conserved methionine‐phenylalanine motif in the core is a unique signature of SUMO but absent in other homologous Ubiquitin‐like (Ubl) protein folds. Using biophysical measurements and solution NMR based experiments, we found that the specific ‘up’ conformation between the methionine‐phenylalanine pair is critical to SUMO’s β‐grasp fold stability and its function(4). Given the additional stability provided by met‐aromatic motif over classical hydrophobic interaction, we engineered new S‐aromatic motifs in SUMO by replacing phenylalanine with other naturally occurring aromatic amino acids. Combining all atom MD simulations and solution NMR studies, we identify the energetically preferred interaction geometry of different met‐aromatic motifs inside the core. We found that the engineered SUMOs with increasing S‐aromatic interaction strength enhances the overall protein stability. Overall, our results reveal the potential role of S‐aromatic interactions as a design tool in enhancing thermodynamic stability of a protein fold.

References

1. Chatterjee, K. S., Tripathi, V., and Das, R. (2019) A conserved and buried edge‐to‐face aromatic interaction in small ubiquitin‐like modifier (SUMO) has a role in SUMO stability and function. J. Biol. Chem. 294, 6772–6784

2. Serrano, L., Bycroft, M., and Fersht, A. R. (1991) Aromatic‐aromatic interactions and protein stability. J. Mol. Biol. 218, 465–475

3. Valley, C. C., Cembran, A., Perlmutter, J. D., Lewis, A. K., Labello, N. P., Gao, J., and Sachs, J. N. (2012) The Methionine‐aromatic Motif Plays a Unique Role in Stabilizing Protein Structure. J. Biol. Chem. 287, 34979–34991

4. Chatterjee, K. S., and Das, R. (2021) An “up” oriented methionine‐aromatic structural motif in SUMO is critical for its stability and activity. J. Biol. Chem. 297, 100970
Microelectrodes for measurement of intracellular [K+] and [Na+]in the choroid plexus epithelium and cerebrospinal fluid
Authors: Berenji Ardestani Simin , Matchkov Vladimir V. , Praetorius Jeppe
Published: May 2022

The choroid plexus epithelium (CPE) has recently gained increasing interest as a potential target tissue to manipulating cerebrospinal fluid (CSF) production (1). It is therefore crucial to identify the basolateral Na+ transport mechanisms that are involved in CSF secretion, as it would be accessible from the blood side and thereby hold promise for pharmacological intervention. The sodium‐bicarbonate transporter Ncbe has been identified as a basolateral Na+ importer in the CPE (2,3). Currently, there is no direct intracellular cation measurements in choroid plexus and this information is needed to establish Ncbe as the main basolateral Na+ importer. The aim of this project is to measure the [Na+] and [K+] concentration in CSF and in CPE in vivo with double‐barreled microelectrodes. Ion selective double‐barreled microelectrodes are the tool to measure directly the different ion concentrations. The reference barreled was filled with 500 mM KCl solution and the tip of ion sensitive barrel was filled with Na+ or K+ionophore and then backfilled with KCl solution. In this ongoing project, we were able to estimate extracellular [K+] to 4.08 ± 1.06 mM in CSF of mice in vivo (n=7). The study is in progress.

References

1: Damkier HH, Brown PD, Praetorius J. Cerebrospinal fluid secretion by the choroid plexus. Physiol Rev. 2013;93:1847‐92.

2: Praetorius J, Nejsum LN, Nielsen S. A SLC4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells. Am J Physiol Cell Physiol. 2004;286:C601‐10.

3: Damkier HH, Nielsen S, Praetorius J. Molecular expression of SLC4‐derived Na+‐dependent anion transporters in selected human tissues. Am J Physiol Regul Integr Comp Physiol. 2007;293:R2136‐46.
Amelioration of Cardiac Autonomic Dysfunction Mitigates Perivascular and Perirenal Adipose Inflammation and Cardiorenal Involvement in Prediabetes
Authors: El‐Yazbi Ahmed F. , Hammoud Safaa , Alzaim Ibrahim F. , Bekdash Amira F. , Mougharbil Nahed , Itani Hana F.
Published: May 2022

Background

Early metabolic impairment is associated with significant cardiorenal risk. Specifically, subtle serum insulin and lipids changes in prediabetes trigger cardioautonomic and renovascular deterioration. Interestingly, data from our laboratory suggest that these insidious alterations are triggered by perirenal and perivascular adipose inflammation in response to hypoxia evoked by a localized increase in uncoupling protein1 (UCP1) expression and activity. This affects the adjacent organs in a paracrine manner.

Hypothesis

Increased UCP1 expression is triggered by sympathetic stimulation. The sympathovagal imbalance in early metabolic dysfunction underlies the observed localized adipose pool involvement. Pharmacological interventions to offset the sympathetic predominance should prevent increased UCP1 expression and inflammation, and reverse cardiorenal manifestations.

Methods

A non‐obese prediabetic rat model was induced by twelve weeks of hypercaloric feeding. Moxonidine and galantamine were administered orally at daily doses of 2.4 mg/Kg and 4 mg/Kg for the last two weeks of feeding to reduce and facilitate sympathetic and parasympathetic discharge, respectively. Baroreflex sensitivity and renovascular reactivity were assessed by invasive hemodynamics and in isolated perfused kidneys, respectively. Adipose tissue T Cell distribution and macrophage polarization were examined by flow cytometry.

Results

Correcting the autonomic imbalance in prediabetic rats with either moxonidine or galantamine restored cardioautonomic and renovascular activity to those observed in control rats. Although prediabetic rats showed an increased oxidative stress and proinflammatory cytokine production in both adipose pools, no change in T Cell population distribution was observed. However, the increased M1/M2 macrophage polarization profile observed in prediabetic rats was reversed by either drug treatment. This was associated with a normalization of UCP1 expression and hypoxic status.

Conclusions

Pharmacological interventions to reverse sympathetic predominance could be of therapeutic value treating perivascular and perirenal adipose inflammation that cause early cardiorenal manifestations of prediabetes.
Different Murine Models of Kidney Injury Reveal a Common Pattern of Dysregulation within the Polyamine System in Favour of its Catabolic Pathways
Authors: Sieckmann Tobias , Ögel Neslihan , Kelterborn Simon , Boivin Felix J. , Schley Gunnar , Fähling Michael , Ashraf Muhammad imtiaz , Reichel Martin , Vigolo Emilia , Hartner Andrea , Knauf Felix , Rosenberger Christian , Aigner Felix , Smidt‐Ott Kai , Scholz Holger , Kirschner Karin M.
Published: May 2022

The polyamines putrescine, spermidine and spermine are organic polycations that regulate many cell functions including proliferation and differentiation. It is known that certain genes of the polyamine system are dysregulated after kidney ischemia reperfusion injury. Here we examined the hypothesis that different forms of acute and chronic kidney injury lead to similar changes in the expression patterns of the polyamine system.

In different models of acute and chronic kidney injury expression of genes involved in polyamine homeostasis were analyzed by RT‐qPCR and RNAScope. In these models, expression of catabolic enzymes (Aoc1 and Sat1) was upregulated, and the anabolic enzymes (Odc1, Sms) were downregulated. The putrescine‐degrading enzyme AOC1 exhibits the most striking changes. Interestingly, it can act together with ODC1 as gatekeepers of the polyamine system. The detected increase of Aoc1 takes place in the injured but regenerating proximal tubules. As a screening for stimuli of increased Aoc1 expression, we used mouse embryonic kidney explants. Here we observed changes of Aoc1expression under hypoxia and hyperosmotic conditions. These changes were further examined in mouse models of hypoxia. However, in vivo, hypoxia did not lead to changes of Aoc1 expression. Hyperosmolarity was confirmed as a stimulus by using the kidney cell lines M15 and 209/MDCT as well as cultured primary proximal tubules. Using reporter gene and RNA‐stability assays, we could show that the increase in Aoc1 expression is based on mRNA‐stabilization and transcriptional activation of one certain isoform. The activated isoform contains an additional set of 22 amino acids N‐terminally that lead to an altered subcellular localization.

In conclusion, different models of kidney injury exhibit a similar pattern of dysregulation of the polyamine system with the most striking change being the upregulation of Aoc1 in proximal tubules. Using hyperosmolarity as a stimulus, we provide first insights into the regulation of Aoc1 under harmful conditions.
Indole‐3‐propionic Acid, Bacterial Metabolite of Tryptophan, Increases Blood Pressure in Normotensive and Hypertensive Rats
Authors: Konopelski Piotr , Chabowski Dawid , Aleksandrowicz Marta , Kozniewska Ewa , Podsadni Piotr , Szczepanska Agnieszka , Ufnal Marcin
Published: May 2022

Tryptophan is an exogenous amino acid, undergoing complex metabolism in mammals, including formation of gut microbiota‐derived metabolites. Tryptophan was proven to increase blood pressure in rats and humans, however; it has not been profoundly investigated, whether its impact on cardiovascular system might be due to the action of microbiota derived indoles, especially indole‐3‐propionic acid (IPA).

Acute hemodynamic experiments were performed on 12‐week‐old Wistar‐Kyoto (WKY) and 16‐week‐old Spontaneously‐Hypertensive (SHR) male anesthetized rats. Mean arterial blood pressure (MABP) and heart rate (HR) were evaluated for 120 minutes after intravenous infusion of 10% DMSO (Vehicle) and IPA in increasing doses (1.6‐53 µM/kg body wt). In separate series of experiments impact of IPA on vascular contractility was evaluated in ex vivo experiments performed on mesenteric resistance arteries (MRAs).

IPA increased significantly blood pressure in normotensive and hypertensive rats. In normotensive rats at highest dose IPA revealed less pronounced hypertensive effect, accompanied by decrease in HR. This effect was not observed in hypertensive rats, where IPA increased MABP in dose dependent matter, with concomitant tachycardic response. In ex vivo experiments IPA increased contractility of endothelium‐denuded MRAs and reduced acetylcholine‐dependent dilatation of MRAs.

Our study suggest, that IPA takes part in regulation of blood pressure, by revealing hypertensive and vasoconstrictive activity. These effects are in line with actions of its precursor tryptophan, suggesting important role of bacterial metabolism of tryptophan in its cardiovascular effects.
Intercellular Ca2+ waves propagate by regenerative IP3‐induced IP3 release
Authors: Buckley Charlotte , Zhang Xun , Lee Matthew , Wilson Calum , McCarron John G.
Published: May 2022

The endothelium is a sheet of highly‐specialised cells that line every blood vessel. Endothelial cells detect, integrate and respond to information from numerous signals, passing this information to neighbouring endothelial cells via intercellular communication to coordinate vascular contractility, inflammation and exchange of products between the blood and surrounding tissues. A significant form of intercellular communication occurs by travelling spatial gradients of Ca2+ (‘Ca2+ waves’) that move between cells. While the underlying mechanisms are not understood, Ca2+ and IP3 diffusion through gap junctions are each proposed to underlie Ca2+ wave propagation in the endothelium. Our results show that IP3, but not Ca2+ or gap junctions, is required for Ca2+ wave propagation.

To investigate the mechanisms underlying endothelial signal propagation, second order mesenteric arteries were dissected from rats, pinned out in an en face configuration and Ca2+ signals measured using Cal‐520 (5μM). Localised photorelease of caged‐IP3 (5μM) generated rapid rises in endothelial Ca2+ at the site of photo‐activation. The Ca2+ rise subsequently propagated across cells outside the photolysis site. The propagated Ca2+ rise reflects cell‐cell communication since no uncaging occurred outside the photolysis site.

To investigate the contribution of Ca2+ to signal propagation, Ca2+ buffering was spatiotemporally controlled using local photoactivation of the membrane permeant Ca2+ buffer, Diazo‐2. Release of the Ca2+ buffer prevented signal propagation within the uncaging region alone; signal propagation still occurred outside the uncaging area. This result suggests that IP3, but not Ca2+, underlies wave propagation. Supportive evidence for this conclusion was provided by photolysis of caged Ca2+(NP‐EGTA AM, 5μM). Photorelease of caged Ca2+ generated a Ca2+ rise that remained localised to the uncaging region and did not propagate outside the photolysis site, even with progressively increased Ca2+ loads. To verify these results, ACh (1μM) or ionomycin (10μM) were applied to restricted populations of cells by pressure ejection from a puffer pipette. A comparable Ca2+ rise was generated in each case, however the ACh generated a substantial Ca2+ wave that propagated upstream of the activated cells while the Ca2+ rise from ionomycin did not. These results suggest that Ca2+ is not responsible for propagating Ca2+ waves in the endothelium. The data also questions the involvement of gap junctions in wave transmission. If gap junctions were present and open, the Ca2+ rise generated by ionomycin would have been expected to diffuse to neighboring cells. This was not observed. Furthermore, the gap junction blockers gap27 and 18αGA, while successful in blocking FRAP, did not inhibit wave propagation. Together, these results suggest that IP3 is essential for Ca2+ wave propagation, but Ca2+ or gap junctions are not.
Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding
Authors: Jay Natalie , Duffy Sean , Tu Jeff , Estrada Fernando
Published: May 2022

The improper maintenance of the bioactivated form of vitamin‐D (1α,25(OH)2D) may result in vitamin‐D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin‐D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon‐23 (C23) or carbon‐24 (C24) of the 1α,25(OH)2D side chain, eventually resulting in production of either an antagonist of the vitamin‐D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilizing hCYP24A1 during production. Subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. The redox partner recognition relies on electrostatics, specifically Asp‐72 and Asp‐76, of hAdx. However, we also demonstrate involvement of a non‐polar contact between Leu‐80 of hAdx and a non‐conserved proximal surface of hCYP24A1. Using a combination of functional assays, spectral binding data, and nuclear magnetic resonance (NMR) spectroscopy, we determine that the contact at Leu‐80 is distance‐dependent; shortening the length of this residue (L80V) results in enhanced binding between the CYP‐Adx complex as measured by protein NMR, promotes more binding of 1α,25(OH)2D, yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24‐hydroxylase), indicating the presence of a species‐specific requirement that may correlate with differences in regioselectivity of the reaction. We combine these findings with an NMR characterization of the hAdx dimer to inform a model of a 1:2 hCYP24A1:hAdx complex.