bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiovascular and Sympathetic Neural Responses to Cognitive Tasks in Women with Posttraumatic Stress Disorder
Authors: Washio Takuro , Yoo Jeung‐Ki , Anderson Elizabeth H. , Takeda Ryosuke , Hissen Sarah L. , Nelson Michael D. , North Carol S. , Suris Alina , Fu Qi
Published: May 2022

Background

Posttraumatic stress disorder (PTSD) is more prevalent in women than men and is associated with an increased risk for dementia. Indeed, we previously showed that cognitive function is impaired in women with PTSD, but the underlying mechanisms remain unknown. In patients with mild cognitive impairment, the cardiovascular response during cognitive tasks have been shown to be blunted. Thus, we hypothesized that cardiovascular and sympathetic neural responses to a cognitive task would be smaller in women with PTSD than healthy women.

Methods

We measured heart rate (HR), mean arterial pressure (MAP), cardiac output (CO, Modelflow), total peripheral resistance (TPR = MAP/CO), and muscle sympathetic nerve activity (MSNA, microneurography) in 16 women with PTSD (42 ± 11 [SD] yrs) and 9 age‐matched healthy women (controls; 41 ± 9 yrs) during 3 min of the Stroop Color and Word Test (SCWT) in the supine position.

Results

In response to SCWT, HR, MAP, and CO increased (all P < 0.001) but TPR remained unchanged (P = 0.753) in both groups. Increases (Δ) in HR (6 ± 4 vs. 11 ± 6 bpm) and CO (0.6 ± 0.6 vs. 0.9 ± 0.4 L/min) during SCWT were smaller in PTSD than controls (both P < 0.05), while ΔMAP was similar between groups (P = 0.169). MSNA burst frequency and incidence were significantly higher in women with PTSD than controls at baseline and throughout SCWT (P = 0.003), but ΔMSNA during SCWT was similar between groups.

Conclusions

Cardiovascular, but not sympathetic neural responses to SCWT were blunted in women with PTSD. Our findings suggest that cognitive impairment in women with PTSD may be related to the insufficient cardiac response rather than sympathetic vasoconstriction during cognitive tasks.
Investigation of selective vagal afferents subserving baroreflex function and blood pressure control: implications for understanding and alleviating hypertension
Authors: Baumer Harrison Caitlin M. , Johnson Dominique N. , Elsaafien Khalid , de Araujo Alan , Penaloza Aponte Jesus , Scott Karen A. , de Lartigue Guillaume , Krause Eric G. , de Kloet Annette D.
Published: May 2022

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors (BRs) that apprise the brain of blood volume and pressure within the carotid sinus and aortic arch. Here, we test the overall hypothesis that neurons within the nodose ganglia that express the angiotensin type 1a receptor (referred to as NDGAT1aR) serve as BR that undergo plasticity during chronic elevations in blood pressure (BP). Towards this end, mice that express Cre‐recombinase at the AT1aR locus were delivered a Cre‐dependent AAV to express tdTomato within NDGAT1aR. These studies revealed that NDGAT1aR receive input from the aortic arch and carotid sinus, project to the caudal nucleus of the solitary tract (cNTS), and synthesize mechanosensitive ion channels, Piezo‐1/‐2. To determine whether NDGAT1aR couple their firing to elevated BP, we generated mice with the expression of the fluorescent Ca2+ indicator, GCaMP6s, directed to the AT1aR locus. Two‐photon (2P) intravital imaging revealed that phenylephrine (PE; i.v.) significantly increased GCaMP6s fluorescence within NDGAT1aR. To determine if optogenetic perturbation of NDGAT1aR at their soma or axon terminals within the cNTS affect the baroreflex, we directed the expression of excitatory (channelrhodpsin2; ChR2) or inhibitory (halorhodopsin; Halo) opsins to the AT1aR locus. Optical excitation of NDGAT1aR at their soma or axons terminals in the cNTS, elicited robust and frequency‐dependent decreases in BP and heart rate. Conversely, optical inhibition of afferents in the cNTS originating from NDGAT1aR blunted pressor responses to PE. The implication is that NDGAT1aR are sufficient and necessary to elicit appropriate compensatory responses to vascular mechanosensation induced by vasoconstriction. Opsin‐ or GCaMP6s‐expressing mice were then subjected to the DOCA‐salt model of hypertension. Optical excitation elicited hypotensive and bradycardic responses; however, the duration of these effects was altered, consistent with hypertension‐induced impairment of BR. Similarly, the increased GCaMP6s fluorescence observed after administration of PE was significantly delayed in mice that were subjected to DOCA‐salt or chronic delivery of angiotensin II. Finally, AT1aR‐flox mice were delivered AAV expressing Cre and/or tdTomato into the nodose ganglia to probe the function of AT1aR(s) themselves, in mediating the baroreflex. Preliminary results indicate deleting AT1aR(s) from the nodose ganglia alters pressor responses to vasoactive drugs. Collectively, these results reveal the structure and function of NDGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.
Kinetic Analysis of T4 Polynucleotide Kinase via Isothermal Titration Calorimetry
Authors: Lim Rebecca C. , Gary Ronald K.
Published: May 2022

Isothermal titration calorimetry (ITC) measures the heat absorbed or released from biochemical processes. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used as a probe to quantify enzyme activity. Kinases are an important class of enzymes that are involved in many types of cellular regulation, but kinetic studies of kinases by ITC have been limited. T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3′‐phosphate group is present. We used ITC to study the T4 PNK‐catalyzed conversion of 3′‐AMP to adenosine‐3′,5′‐bisphosphate. To initiate the reaction, a single injection of 3′‐AMP substrate was made into the ITC cell containing T4 PNK and ATP at pH 7.6 and 30oC. The shape of the resultant Differential Power versus Time curve is determined by the kinetic properties of the enzyme. Integration of the area under the curve yields reaction enthalpy. The apparent ∆H for the reaction was found to vary depending on the type of buffer used. This result was explained by differential protonation of reactants and products at pH 7.6, and indicated the release of approximately 0.6 protons per mole for the forward reaction. Extrapolation was used to estimate an intrinsic (i.e. buffer‐independent) ∆H of ‐25 kJ/mol. The Michaelis‐Menten constant, KM, was 1.3E‐05 M, and the turnover number, kcat, was 8/sec. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3′‐AMP were assessed successfully by single injection ITC analysis. This finding supports the potential to analyze more complex kinase systems using ITC.
Autophagic Deficiency and Dedifferentiation in Podocytes of Mice Lacking Acid Ceramidase (Asah1) Gene
Authors: Li Guangbi , Huang Dandan , Zou Yao , Li Ningjun , Ritter Joseph , Li Pin‐Lan
Published: May 2022

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain with a podocyte‐specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Asah1fl/fl/Podocre, Asah1 is a gene code of mouse acid ceramidase). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether autophagic deficiency and associated dedifferentiation of podocytes contribute to the pathogenesis of podocytopathy in Asah1fl/fl/Podocre mice. By Western blot analysis and immunofluorescent staining, we demonstrated the significant reduction of epithelial markers (ZO‐1 and P‐cadherin) and remarkable elevation of mesenchymal markers (α‐SMA and FSP‐1) in podocytes lack of Asah1 gene in vivo and in vitro. Also, we found that Asah1 gene knockout induced autophagic deficiency in podocytes, which were evidenced by autophagosome accumulation and decreased lysosome‐autophagosome interaction in these cells. Given the vital role of transient receptor potential mucolipin 1 (TRPML1) channel in the regulation of lysosome trafficking and interaction with other vehicles, we tested whether this lysosomal channel is implicated in autophagic deficiency in podocytes lacking Asah1 gene. As a selective TRPML1 channel agonist, ML‐SA5 enhanced lysosome‐autophagosome interaction in WT/WT podocytes, but only had partial effects on this interaction in Asah1fl/fl/Podocre podocytes. Autophagosome accumulation was also attenuated by ML‐SA5 in podocytes lacking Asah1 gene. On the contrary, ML‐SI1 as a TRPML1 channel inhibitor impaired autophagic flux in WT/WT podocytes and amplified autophagic deficiency in Asah1fl/fl/Podocre podocytes. The enhancements of lysosome trafficking and lysosome‐autophagosome interaction by ML‐SA5 were blocked by dynein inhibitors, EHNA and ciliobrevin D, indicating the contribution of dynein to lysosome movement in response to TRPML1 channel‐mediated Ca2+ release. Pathologically, Asah1 gene deletion‐induced podocyte dedifferentiation, reduction of slit diaphragm proteins, and proteinuria were inhibited by ML‐SA5 but enhanced by ML‐SI1. Based on these results, we conclude that Asah1 gene deletion induces autophagic deficiency and dedifferentiation in podocytes through blockade of lysosomal TRPML1 channel, thereby leading to the development of podocytopathy and nephrotic syndrome in Asah1fl/fl/Podocre mice.
Intestinal AMPK modulation of gut microbiota mediates cross‐talk with brown fat to control metabolism
Authors: Huang Wendong , Zhang Eryun , Jin Lihua , Wang Yangmeng , Fang Zhipeng , Al‐Abdullah Ismail , Shuck Sarah
Published: May 2022

The energy‐dissipating capacity of brown adipose tissue through thermogenesis can be targeted to improve energy balance. Mammalian 5′‐AMP‐activated protein kinase (AMPK), a key nutrient sensor for maintaining cellular energy status, is a known therapeutic target for glucose control in Type II diabetes (T2D). Despite current understandings of its well‐established roles in regulating glucose metabolism in various tissues, the functions of AMPK in the intestine, an organ for nutrient processing, remain largely unexplored. In this study, we generated the intestinal AMPKα1 specific knock out mice (IKO) and observed that the AMPKα1 IKO mice displayed impaired thermogenesis function of brown adipose tissue both in chow diet and high fat diet condition. Moreover, HFD‐fed AMPKα1 IKO mice showed more severe impaired glucose tolerance and insulin resistance. Mechanistically, we revealed that intestinal AMPK can regulate the anti‐microbial peptides (AMPs) Reg3 and result in a shift in the gut microbiota composition, which leading to the elevation of the fermentation products methylglyoxal, and methylglyoxal can significantly suppress the UCP1 expression in the brown adipose tissue. While transplantation of the microbiota from AMPKα1 IKO mice to antibiotic‐induced microbiome depletion (AIMD) mice, the mice also showed impaired thermogenesis function of brown adipose tissue and elevated serum methylglyoxal level. In addition, we found that the metabolic effect and the AMP regulation of metformin is dependent on the intestinal AMPK.

These results demonstrated that the intestinal nutrient sensor AMPKα1 can regulate the thermogenesis function of brown adipose tissue through modulating AMKP expression and reshaping the gut microbiome. These findings provide a new gut‐brown adipose tissue axis mechanism for activating energy expenditure and treating metabolic diseases.
Pressure Only Wave Separation Pulsatile Hemodynamics in Adolescents: Accuracy and Associations with Left Ventricular Mass Index
Authors: Armstrong Matthew K. , Chirinos Julio A. , Kapuku Gaston K. , Pierce Gary L.
Published: May 2022

Introduction

Early‐life exposure to cardiovascular disease (CVD) risk factors, such as high blood pressure (BP), is associated with cardiovascular target organ damage in later life. However, not all BP‐related risk is explained by higher systolic and diastolic BP alone. In adolescence, aortic wave separation (WS) pulsatile hemodynamic parameters are associated with adverse left ventricular (LV) structure but are limited by the requirement for measured aortic flow. Several methods for estimating aortic flow from pressure waveforms have emerged, but their accuracy and associations with LV structure have never been tested in adolescents. We hypothesized that pressure‐only estimated flow waveforms would: 1) accurately approximate measured flow hemodynamic WS parameters and 2) be similarly associated with LV mass as measured flow hemodynamics among healthy adolescents.

Methods

. BP waveforms were acquired via carotid tonometry in 58 adolescents (age 16±1.5 years, 59% females). Measured (aortic) flow and LV mass index (LVMI) were measured via 2D echocardiography. Three pressure‐only approximations of aortic flow waveforms were synthesized from analysis of the BP waveform, including triangular, excess, and individualized physiologic flow. A 4th aortic flow waveform (average flow) was approximated from the average of all 58 measured flow waveforms. Forward (Pf) and backward (Pb) pressure and reflection magnitude (Rm) were derived from WS analysis.

Results

. Compared to measured flow, individualized physiologic and average flow produced the best approximations of Pf (mean difference ± SD, ‐0.15±2.38 and 1.23±2.84 mmHg respectively), Pb (0.14±0.25 and 0.67±0.10 mmHg), and Rm (0.01±0.02 and ‐0.01±0.02 mmHg). Pf derived using all flow waveforms, measured, and estimated, was associated with LVMI (R >0.44, P <0.001 for all), and these associations yielded similar effect estimates in all instances (z‐score <1.1, P >0.28). All associations persisted after adjusting for age, systolic BP, heart rate, and aortic diameter (P <0.05 all).

Conclusion

. Of the estimated flow waveforms, individualized physiologic flow yielded the best approximation of WS hemodynamic parameters. It may provide important physiological and clinical insight related to cardiovascular target organ damage among adolescents.
Linoleic Acid‐Derived Diol 12,13‐DiHOME Modulates THP‐1 Macrophage NLRP3 Inflammasome Activation
Authors: Valencia Robert , Azer Brandon , Seubert John
Published: May 2022

While inflammatory dysregulation is a major contributor to cardiac injury and worsened functional recovery, current anti‐inflammatory therapies have shown limited success, necessitating studies which identify gaps in our mechanistic understanding of inflammation in the heart. Essential dietary N‐3 and N‐6 polyunsaturated fatty acids (PUFAs) are metabolized into a family of PUFA epoxides and downstream diols with contrasting effects in cardiac inflammatory disease, suggesting metabolite‐ and cell‐specific roles for these molecules. Previous studies have demonstrated 12,13‐dihydroxyoctadecenoic acid (12,13‐DiHOME), a linoleic acid‐derived diol, mediates mitochondrial damage and inflammation in cardiomyocytes, though its roles in other cardiac cell types are unknown. We hypothesized that 12,13‐DiHOME enhances macrophage inflammation by promoting M1 polarization and NLRP3 inflammasome activation. To study macrophage polarization, PMA‐differentiated THP1 “M0” macrophages were incubated with vehicle, 12,13‐DiHOME (0.5 µM), M1‐polarization stimuli (10 ng/mL LPS and 20 ng/mL IFN‐gamma) or M1‐polarization stimuli + 12,13‐DiHOME for 24 hours. Using quantitative real‐time PCR we observed that 12,13‐DiHOME alone did not induce M1 polarization but M1‐associated cytokine TNF gene expression was enhanced in macrophages treated with 12,13‐DiHOME alone or M1‐polarization stimuli + 12,13‐DiHOME. The NLRP3 inflammasome response was activated in THP1 M0 macrophages by first priming with LPS (10 ng/mL) for 4.5h followed by treatment with nigericin (10 µM) for 30min. NLRP3 inflammasome activation was assessed by measuring key markers (NLRP3, caspase‐1, interleukin‐1β). We observed a concentration‐dependent increase in NLRP3 inflammasome activation when 12,13‐DiHOME was co‐treated with LPS‐priming. Next, we used epifluorescence microscopy to assess mitochondrial dysfunction, which is known to modulate macrophage NLRP3 inflammasome activation, by measuring alterations in membrane potential with TMRE (50nM). Time course analyses demonstrated no differences in mitochondrial membrane potential between LPS‐primed and LPS+12,13‐DiHOME‐primed macrophages prior to nigericin‐activation. However, LPS+12,13‐DiHOME‐primed macrophages activated with nigericin had a more rapid decline in membrane potential compared to nigericin‐activated macrophages primed with LPS alone, suggesting exaggerated mitochondrial damage. Overall, our data demonstrates 12,13‐DiHOME does not trigger macrophage polarization alone but enhances macrophage NLRP3 inflammasome activation potentially by causing mitochondrial injury.
Outer Membrane Regulation in Acinetobacter: Controlling the Shield against Antibiotics
Authors: Brown Ashley , Raustad Nicole , Geisinger Edward
Published: May 2022

Acinetobacter baumannii is a bacterium the CDC categorizes as an urgent threat due to its distinctive level of antibiotic resistance. In A. baumannii, BfmRS is a global regulatory system that controls much of this resistance through regulating the cell envelope of the bacterium. However, the mechanisms with which BfmRS controls envelope synthesis and antibiotic resistance are unclear. Whether BfmRS regulates certain outer membrane proteins to control antibiotic resistance was studied. To test this hypothesis, reporters of gene expression of two outer membrane proteins, Omp25 and CarO, were constructed and then using the fluorescent protein GFP, the effect of BfmRS activating or inactivating mutations on the strength of gene expression was measured. The two genes were also deleted from the bacterial chromosome, and the resulting change in resistance to colistin, an important “last line of defense” drug against A. baumannii, were determined. From these experiments, the outer membrane proteins were indeed found to be regulated by BfmRS and to contribute to intrinsic colistin resistance. These findings suggest that Omp25 and CarO are important targets of regulation by BfmRS related to antibiotic resistance. Potential binding motifs in their promoter regions that may determine this regulation were also identified. For next steps, we will examine the role of these motifs on the strength of BfmRS‐dependent gene expression by testing truncated promoters with fewer predicted BfmRS target sites. The binding motifs will also be modified through substitution mutations and then effects on gene expression levels will be measured. The effect of Omp25 and CarO on permeability to additional antibiotics and molecules will be tested. Altogether, this work offers insight into the mechanisms of BfmRS control of the cell envelope and drug resistance, which can help us use the system as a potential target for new treatments.
Inhibition of Phosphoinositide 3‐kinase delta (PIK3CD) Suppresses Hepatocyte Proliferation by More than 50% in the Regenerating Liver after Partial Hepatectomy
Authors: Martucci Nicole J. , Stoops John W. , Bowen William C. , Mars Wendy M. , Michalopoulos George K.
Published: May 2022

Background and Objective

The extracellular matrix (ECM) is important for survival, differentiation, and normal functioning of cells within the liver; integrins are key signaling receptors in this process. Previous data from our lab has shown that with hepatocyte specific knockout of integrin linked kinase (hILK KO), there is hepatocyte proliferation, increased matrix deposition, and unorganized biliary cell/ductal proliferation; this led us to investigate the signaling pathways downstream of hILK KO that might be responsible for the observed phenotype. Through this, we uncovered a possible central role of Phosphoinositide 3‐kinase (PI3K) delta (PIK3CD), a protein thought to be immune specific and absent in hepatocytes. The objective of this study was to determine the role PIK3CD has in the liver. From literature searches and our initial data collection, our hypothesis was that the inhibition of PIK3CD would suppress hepatocyte proliferation.

Methods

Data array analyses were performed on livers from 7‐ and 14‐week old chronic hILK KO and WT mice. Partial Hepatectomies (PHx) were performed on C57BL/6J mice injected IP with vehicle control or the PIK3CD inhibitor, Cal‐101, at 10mg/kg, two days prior to PHx, and every day following until harvest. Days 0, 1, 2, 4 and 7 post‐surgery were examined through immunohistological and western blot analyses. RNA was generated and pooled (n=6) for each condition and timepoint and sent out for analyses. Additionally, in vitro cell culture was performed using primary hepatocytes isolated from hILK WT mice. Hepatocytes were seeded at 300K cells per well, with media changed every other day and treated with or without 5uM CAL‐101 over the course of 6 days. BrdU was added 2 hours prior to harvest of cells to assess proliferation.

Results

Data array analyses on ILK KO and WT animals by IPA identified PIK3CD as common to several pathways in the ILK KO mice. WT mice that were treated with the PIK3CD inhibitor, Cal‐101, exhibited a significant decrease in hepatocyte proliferation on days 1, 2, 4, and 7 after PHx, compared to vehicle control, through quantification of nuclear Ki67 staining. By day 7, proliferation subsided in both the control and Cal‐101 mice and liver to body weight ratios were similar. Additionally, western blot analyses revealed differences in p‐AKT and p‐ERK on days 0,1, and 2 after PHx, as well as changes in p‐MET at days 0 and 1. In vitro culture data also supported a role for PIK3CD in hepatocyte proliferation as a significant decrease was observed in primary hepatocytes treated with Cal‐101 compared to control over a 6‐day culture period.

Conclusion

This data shows a previously unknown essential role for PIK3CD in controlling hepatocyte proliferation in the regenerating liver.
Selective Transport in the Nuclear Pore Complex
Authors: Cowburn David
Published: May 2022

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic exchange controlling the flow of molecules into and out of the nucleus. The selective filter properties of NPCs enable translocation of specific molecules known as nuclear transport factors (NTRs) and their cargo. Central to this selectivity barrier is a group of largely intrinsically disordered nucleoporins (Nups) that contain multiple phenylalanine‐glycine repeats, termed FG Nups. The interactions between FG Nups and NTRs enable NTRs to translocate rapidly yet selectively through the NPC in what is referred to as the “transport paradox”. FG Nups in the NPC have a small folded anchor region tethering them to the NPC outer ring and otherwise are fully disordered, random coil polymers that remain predominantly disordered while engaged to NTRs. FG Nups interact with NTRs using mainly their FG motifs and minimally their intervening spacer residues. The overall enthalpy of the interaction increases as multivalency increases the frequency of individually weak FG‐NTR contacts. Tight binding is limited by an entropy penalty that disfavors simultaneous engagements of FG motifs. Small angle neutron scattering (SANS) shows that the entropy loss is partly due to the local rigidity of an FG motif in the interacting state(Fig 1). All‐atom molecular dynamics (MD) simulation indicates that spacers between the FG motifs behave as “entropic springs”, disfavoring any static binding of the FG repeats. The dynamics of FG Nups enables the FG motifs to slide along the hydrophobic patches of NTRs enabling FG motifs to be easily displaced by other competing FG motifs (Fig. 2). This explanation provides a simple hypothesis for the rapid exchange of FG motif contacts during transport, focusing on the entropic exclusion of non‐NTRs, and ‘solubilization’ of NTR complexes, in contrast to possible condensate formation which would provide a compartmentalization of components. These results reveal fundamental aspects of the functioning mechanisms underlying NPC transport at high structural resolution, something lacking in current models of nuclear transport.

1. Sparks, S., et al., Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements. Methods Mol Biol, 2020. 2141: p. 463‐475.

2. Sparks, S., et al., Deciphering the “Fuzzy” Interaction of FG Nucleoporins and Transport Factors Using Small‐Angle Neutron Scattering. Structure, 2018. 26(3): p. 477‐484 e4.

3. Hayama, R., et al., Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex. J Biol Chem, 2018. 293(12): p. 4555‐4563.

4. Hough, L.E., et al., The molecular mechanism of nuclear transport revealed by atomic‐scale measurements. Elife, 2015. 4.

5. Raveh, B., et al., Slide‐and‐exchange mechanism for rapid and selective transport through the nuclear pore complex. Proc Natl Acad Sci U S A, 2016. 113(18): p. E2489‐97.
Sodium Status Modulates the Effects of Aldosterone on Renal Transporter Expression and Tubular Remodeling
Authors: Mutchler Stephanie M. , Baty Catherine J. , Kleyman Thomas R.
Published: May 2022

Aldosterone (aldo) is a steroid hormone that has a key role in extracellular volume and K+ homeostasis. While aldo levels are expected to be suppressed with high Na+intake, 5‐10% of hypertensives have chronic hyperaldosteronism regardless of Na+ intake. This combination of high sodium and high aldo can worsen end organ damage, especially in the kidney. We examined the effects of elevated aldo in the absence or presence of a high salt diet (HSD) on the expression of Na+ transporters in the distal nephron and on distal nephron remodeling to understand if these changes may play a role in the altered pathology. Male C57Bl/6 mice received either a control (0.4% NaCl) or HSD (4 weeks, 8% NaCl), +/‐ aldo via minipump for the final 2 weeks of treatment (240 μg/kg/day). Transporter expression levels and localization were analyzed by Western blot, qPCR and immunofluorescent staining. Expression of the α subunit of the epithelial sodium channel (ENaC) was increased with aldosterone regardless of Na+ intake, while subunits increased at both a transcript and protein level in mice receiving aldo and HSD. Both total levels of sodium chloride cotransporter (NCC) and active pNCC significantly increased with aldo alone, in association with a fall in blood [K+]. However, the addition of HSD markedly attenuated this increase in NCC and pNCC, despite more severe hypokalemia. This change was not associated with a difference in the expression of its upstream kinase SPAK. NKCC2 expression was not significantly altered in any of the conditions. We imaged whole tubules in thick kidney slices (300μm) to assess whether remodeling of the distal tubule could account for NCC expression differences. Sections were incubated with antibodies against pNCC and AQP2 and cleared with ethyl cinnamate to allow for imaging and analysis of whole distal tubules. Length of the distal tubule was analyzed as the pNCC positive portion. While aldo has previously been shown to promote hypertrophy of the distal tubule, we found a significant shortening of tubules in animals treated chronically with 2 weeks of aldo (Ctl 434m ± 63μm versus aldo 375m ± 88m, P<0.05, n=38 and 30 respectively). However, the addition of HSD with aldo caused a significant increase in distal tubule length (486m ±68μm, P<0.05, n=23). HSD by itself did not alter distal tubule length (428m ± 55m, n=17). Our results suggest that aldo‐mediated transcriptional regulation of ENaC subunits in the kidney changes according to dietary Na+ status. The attenuation of increased NCC and pNCC expression in kidneys of mice receiving HSD with aldo, despite profound hypokalemia and an increase in DCT length, suggest that factors other than blood [K+] have important roles in regulating NCC expression. Finally, aldo‐dependent DCT remodeling is dependent on dietary Na+.
Does Dietary Methionine Alter Perivascular Adipose Tissue Function?
Authors: Lee Davis , Corken Adam , Wahl Elizabeth C. , Thakali Keshari M.
Published: May 2022

Perivascular adipose tissue (PVAT) is an adipose depot surrounding blood vessels and is a paracrine regulator of vascular tone. Obesity is associated with the loss of PVAT‐mediated anti‐contractile properties, thus contributing to obesity‐associated hypertension. Hydrogen sulfide (H2S) is a gaseous signaling molecule that is produced in the vasculature and is important for regulating vascular tone. In PVAT, cystathionine gamma lyase (Cse) is the primary source of H2S which acts as an important adipose‐derived relaxing factor. Dietary methionine restriction recapitulates many of the metabolic effects of caloric restriction and leads to preferential expression of activating transcription factor 4 (ATF4) which facilitates Cse transcription. We hypothesized that dietary methionine restriction will prevent high fat diet‐induced loss of PVAT anti‐contractile properties, through activation of the integrated stress response (ISR) and increased ATF4‐mediated transcription of Cse. Male Wistar rats were fed either a control (C, 10% calories from fat) or a high fat diet (HFD) (H, 60% calories from fat) that was either methionine replete (R, 0.86% methionine) or methionine depleted (D, 0.12% methionine) for 12 weeks such that there were four groups: CR, CD, HR, and HD. Dietary methionine restriction prevented body weight gain, while rats fed a methionine replete diet gained weight over the 12 week feeding period. There was no difference in weight gain between control or HFD fed rats. Arterial blood pressure, measured in conscious rats using tail cuff plethysmography, was not different between the four groups over the 12 week feeding period. After 12 weeks on diets, arterial contractility was assessed using wire myography in 2nd order mesenteric arteries (MAs) with and without attached PVAT. In our hands, the presence of PVAT was associated with reduced contraction to norepinephrine (NE, 10 µM) and greater relaxation to acetylcholine (ACh,10 µM) in MAs from all groups, and HFD was not associated with a loss of PVAT anti‐contractile properties. In the presence of PVAT, dietary methionine restriction increased relaxation to ACh in NE‐precontracted MAs, while HFD had no effect on ACh‐induced relaxation. In conclusion, in male Wistar rats, while HFD had no effect on weight gain, blood pressure, or PVAT modulation of MA contractility, dietary methionine restriction for 12 weeks had profound effects on body weight gain, no effect on arterial blood pressure, and a small effect on MA ACh‐induced relaxation in the presence of PVAT. Future studies include examining gene and protein expression of Cse and other ATF4 responsive genes using real time qPCR, Western blot analysis, and immunohistochemical staining of arterial cross‐sections. These studies will provide further insights into dietary interventions to minimize the cardiovascular co‐morbidities associated with obesity.
Investigation into the Role of the Skeletal Muscle BBSome in Insulin Sensitivity
Authors: Rouabhi Younes , Guo Deng‐Fu , Rahmouni Kamal
Published: May 2022

The BBSome is a protein complex composed of eight Bardet‐Biedl syndrome (BBS) proteins including BBS1. Humans and mice lacking a functional BBSome display impaired insulin sensitivity, hyperglycemia, and type 2 diabetes, highlighting the importance of this protein complex in the control of insulin action and glucose homeostasis. However, the contribution of the BBSome in insulin sensitive tissues such as the skeletal muscle to the control of insulin sensitivity is not clear. We hypothesized that BBSome deficiency in the skeletal muscle will lead to insulin resistance and diabetes. To test this, we generated a novel conditional knockout mouse model lacking the Bbs1 gene specifically in the skeletal muscle. This was achieved by crossing mice expressing floxed alleles of the Bbs1 gene (Bbs1fl/fl) with mice expressing Cre‐recombinase in the skeletal muscle (MCKCre). These mice were further bred with tdTomato reporter mice to validate Cre‐recombinase using red tdTomato protein fluorescence. Body weight, glucose handling (measured using glucose tolerance test, GTT) and insulin sensitivity (measured using insulin tolerance test, ITT) of Bbs1fl/fl/MCKCre mice were compared to littermate controls. Furthermore, we used Western blot to test the ability of insulin to activate Akt, a key signaling pathway associated with the insulin receptor. We found that both male and female Bbs1fl/fl/MCKCre mice have no significant alteration in body weight relative to wild type controls (31.4±1.5 vs 31.1±1.0 g in males and 24.1±2.2 vs 23.0±1.7 g in females at 16 weeks of age). There was also no significant change in glucose handling (GTT AUC: 30608±2106 vs 30741±3480 in males and 28836±4466 vs 26530±3334 AUC in females) and insulin sensitivity (ITT AUC: 11017±1285 vs 9935±1080 in males and 8223±792 vs 5803±978 AUC in females) between Bbs1fl/fl/MCKCre mice and controls. This was further confirmed by the lack of difference in insulin‐induced Akt activation in skeletal muscle, liver, and adipose tissue. These findings suggest that BBS1 protein in skeletal muscle is not required for insulin sensitivity. Next, we assessed whether challenging the mice with high fat/high sucrose diet (HFHSD), starting at weaning, could reveal a role for the skeletal muscle BBSome in insulin sensitivity. The weight gain evoked by HFHSD was comparable between Bbs1fl/fl/MCKCre mice and controls (32.3±3.1 vs 33.3±1.0 g in males at 10 weeks of age). Once they reach 16 weeks of age, the HFHSD mice will be subject to glucose tolerance and insulin sensitivity tests to determine if obesity exaggerates hyperglycemia and insulin resistance in Bbs1fl/fl/MCKCre mice.
Intra and inter‐day reproducibility of the functional sympatholysis protocol during rhythmic handgrip exercise in healthy humans
Authors: Teixeira André L. , Garland Matthew , Lee Jordan B. , Nardone Massimo , Millar Philip J.
Published: May 2022

Introduction

The effects of increased sympathetic activity on vasoconstriction are blunted in active skeletal muscle during exercise. This mechanism, termed functional sympatholysis (FSY), plays a vital role in the maintenance of blood perfusion and oxygen delivery to meet the metabolic demands of active skeletal muscle. In humans, FSY can be determined experimentally by applying low‐levels of lower‐body negative pressure (LBNP) to unload the cardiopulmonary baroreceptors and induce a reflex sympathoexcitation at rest and simultaneously during rhythmic handgrip exercise. In young healthy individuals, LBNP causes a pronounced vasoconstrictor response at rest, but not during exercise in the working forearm. Although this paradigm has been used extensively to assess the FSY in humans, to date, no studies have examined the reproducibility of this protocol. Therefore, the aim of the present study is to assess the within‐visit and between‐visit reproducibility of the FSY protocol in healthy adults.

Methods

Twenty‐three (12 females) young, healthy adults (26 ± 4 years; 73 ± 13 kg [mean ± SD]) participated in the study. Forearm blood flow (Doppler ultrasound) and beat‐to‐beat BP (finger photoplethysmography) were measured continuously at rest and during 6‐min of rhythmic handgrip exercise at 30% of maximum voluntary contraction. LBNP at ‐20 mmHg was applied for 2 min at rest and from the 3rd to the 5th min of handgrip. Participants performed two visits separated by ≥48h where two FSY bouts were completed. Forearm vascular conductance (FVC) was calculated as FBF/Mean blood pressure. The FSY index (FSYi) was calculated using the following formula: FSYi = (∆%handgrip ‐ ∆%rest)/100. Intraclass correlation coefficients (ICC; two‐way mixed effects, average measures, and absolute agreement) were used as a primary reproducibility measure (ICC values >0.75 are considered very good).

Results

At rest, LBNP induced a decrease in forearm blood flow (Visit 1 trial 1: ∆‐43±16%; Visit 1 trial 2: ∆‐38±10%; Visit 2: ∆‐39±14%) while these responses were attenuated during LBNP+handgrip (Visit 1 trial 1: ∆‐7±7%; Visit 1 trial 2: ∆‐5±8%; Visit 2: ∆‐4±7%). Each of these responses was similar across the trials (all P≥0.17). The FSYi calculated based on the forearm blood flow responses demonstrated excellent intra (ICC=0.93 [95% CI: 0.82‐0.97]; P<0.001) and inter‐day (ICC=0.94 [95% CI: 0.85‐0.97]; P<0.001) reproducibility. Similarly, FVC responses demonstrated very good intra (ICC=0.81 [95% CI: 0.55‐0.92]; P<0.001) and inter‐day (ICC=0.86 [95% CI: 0.65‐0.95]; P<0.001) reproducibility.

Conclusion

This is the first study to demonstrate that Doppler ultrasound‐derived assessments of FSY using LBNP and forearm handgrip exercise possess high intra‐ and inter‐day reproducibility in young, healthy adults.
Attenuation of Obesity‐Induced Glomerular Inflammatory Response by Inhibition of Exosome Biogenesis and Release without Changes in NLRP3 Inflammasome Activation in Podocytes
Authors: Huang Dandan , Li Guangbi , Zou Yao , Li Ningjun , Ritter Joseph K. , Li Pin‐Lan
Published: May 2022

Obesity has been reported to induce NLRP3 inflammasome activation in podocytes, leading to podocyte injury and glomerular dysfunction. The present study tested whether exosome biogenesis and lysosome function are involved in the regulation of exosome release from podocytes during obesity and whether obesity still induces glomerular inflammatory response without robust release of exosomes. By nanoparticle tracking analysis, increased urinary exosome excretion was found in WT/WT mice fed with high‐fat diet (HFD), which was exaggerated by podocyte‐specific overexpression of Smpd1 gene (Smpd1 is the gene code of mouse acid sphingomyelinase) in Smpd1trg/Podocre mice. Exosome biogenesis inhibitor‐GW4869, lysosome function enhancer‐rapamycin, and acid sphingomyelinase inhibitor‐amitriptyline were found to inhibit the elevation of urinary exosome excretion during obesity in both WT/WT and Smpd1trg/Podocre mice. We also isolated podocytes from WT/WT and Smpd1trg/Podocre mice for in vitro studies. Visfatin as a pro‐inflammatory adipokine enhanced exosome release from podocytes, which was exaggerated by Smpd1 gene overexpression. Correspondingly, pre‐treatment of cells with GW4869, rapamycin, and amitriptyline blocked the increase in exosome release induced by visfatin. By confocal microscopy, we observed HFD‐induced NLRP3 inflammasome activation in glomeruli, which was enhanced by podocyte‐specific Smpd1 gene overexpression but attenuated by amitriptyline. Moreover, HFD‐induced immune cell infiltration into glomeruli and glomerular sclerotic changes were worsen by podocyte‐specific Smpd1 gene overexpression but inhibited by all 3 treatments. However, GW4869 and rapamycin had no effects on NLRP3 inflammasome in podocytes, but only amitriptyline suppressed NLRP3 inflammasome activation. It is concluded that exosome release from podocytes during obesity contributes to the inflammatory response in glomeruli, which is regulated by exosome biogenesis and lysosome function. Without robust release of exosomes, NLRP3 inflammasome activation may not induce glomerular inflammatory response during obesity.
A Neuromodulatory System that Links Opioid‐Induced Sedation and Respiratory Depression
Authors: Varga Adrienn G. , Maletz Sebastian N. , Dossat Amanda M. , Reid Brandon T. , Levitt Erica S.
Published: May 2022

The main cause of death from opioid overdose is respiratory depression, mediated by mu‐opioid‐receptors located in the respiratory brainstem. Opioids also cause sedation, which further reduces respiratory drive through largely unknown mechanisms. The Kölliker‐Fuse nucleus (KF) in the pons is highly susceptible to inhibition by opioids and significantly contributes to opioid‐induced respiratory rate depression and associated changes in breathing pattern. Taking advantage of cell‐type specific retrograde neural tracing, we characterized anatomical projections between the main noradrenergic arousal center of the brain, the locus coeruleus (LC) and the KF, which represent a potential link between arousal/sedation and breathing. Our results show that 30% of noradrenergic LC neurons send projections to the KF. To begin to understand the functional role and to define the neurochemical identity of LC to KF projections, we used whole‐cell patch clamp recordings from the KF combined with optogenetic stimulation of LC neurons. Optical stimulation of LC axon terminals induces light‐evoked glutamatergic excitatory postsynaptic currents in KF neurons. Pharmacological isolation of LC to KF synapses suggests that this pathway is monosynaptic. The glutamatergic excitatory postsynaptic currents are dependent on vesicular glutamate transporter 2 expression in noradrenergic LC neurons. Additionally, prolonged optical stimulations lead to noradrenergic excitatory postsynaptic currents that are blocked by alpha1 receptor antagonist. Thus, LC may drive KF neuron activity via glutamate and/or noradrenaline release. This direct excitatory drive in the LC‐KF circuit is sensitive to opioids, which can directly inhibit KF neuron somas, as well as inhibit neurotransmitter release from presynaptic LC terminals. This work provides exciting evidence for an excitatory pathway between the LC and the KF, that may play an important link between opioid‐induced sedation and respiratory depression.
Impact of Maternal Obesity on Resting Muscle Sympathetic Nerve Activity during Normotensive Pregnancy
Authors: Hissen Sarah L. , Takeda Ryosuke , Yoo Jeung‐Ki , Badrov Mark B. , Tomlinson Andrew , Khan Safia , Nelson David B. , Babb Tony G. , Fu Qi
Published: May 2022

Maternal obesity increases the risk of adverse pregnancy outcomes. The mechanisms that contribute to this elevated risk are unclear but may be related to overactivation of the sympathetic nervous system. Indeed, sympathetic activation during pregnancy is now regarded as a normal adaptation to maintain homeostasis. However, sympathetic overactivation is associated with gestational hypertensive disorders. It is unknown whether the health related problems linked to maternal obesity (i.e., inflammation, insulin resistance, dyslipidemia) results in sympathetic overactivation during pregnancy. We hypothesized that maternal obesity would induce greater increases in resting muscle sympathetic nerve activity (MSNA) during pregnancy when compared with normal‐weight women.

Blood pressure (BP), heart rate and MSNA (microneurography) were recorded during 5 min of supine rest in 14 normal‐weight (Age 30±1.6 [SE] yrs; BMI 22.1±0.6 kg/m2) and 14 obese (Age 28±0.8 yrs; BMI 33.9±1.0 kg/m2) women during early pregnancy (5‐12 weeks), late pregnancy (32 to 34 weeks) and 6‐10 weeks postpartum.

Gestational weight gain from early to late pregnancy was not significantly different between groups (11.8±0.9 kg vs. 9.2±2.1 kg, P=0.23). Resting MSNA burst frequency was not different between normal‐weight and obese groups during early pregnancy (17±3 bursts/min vs. 22±4 bursts/min, P=0.35) or postpartum (10±2 bursts/min vs. 8±2 bursts/min P=0.74) but was significantly greater in the obese group during late pregnancy (23±4 bursts/min vs. 35±4 bursts/min, Figure A, P=0.031). There were no significant changes in resting MSNA from early to late pregnancy in the normal‐weight group (P=0.67) whereas the obese group had significant increases in MSNA from early to late pregnancy (P=0.002). These findings were also apparent when MSNA was characterized as burst incidence and total activity (Figure B and C). Although still within the normotensive range, women with obesity had greater levels of systolic BP when compared with normal‐weight women across all time points (Figure D, group main effect P=0.002). Diastolic BP was lower during pregnancy when compared with postpartum and this was similar between groups (Figure E, gestation main effect P<0.001). Heart rate was greater during early pregnancy when compared with postpartum and increased similarly in both groups during late pregnancy (Figure F, gestation main effect P<0.001).

These findings suggest that maternal obesity is associated with greater increases in sympathetic activation even during normotensive pregnancy and may play a role in the increased risk of adverse maternal and fetal outcomes in women with obesity.
P‐cadherin regulates intestinal epithelial repair, but is dispensable for colitis associated colon cancer development
Authors: Naydenov Nayden G. , Lechuga Susana , Zalavadia Ajaykumar , Huang Emina , Ivanov Andrei I.
Published: May 2022

Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases (IBD), triggers significant changes in the intestinal epithelial homeostasis. These changes include leakiness of the gut barrier, formation of mucosal wounds and, in most severe cases, oncogenic transformation of colonic epithelium resulting in colitis‐associated colon cancer (CAC). Altered structure and dynamics of epithelial junctions is a hallmark of intestinal inflammation, mediating epithelial barrier injury and repair. P‐cadherin (gene name: CDH3) is an important component of adherens junctions (AJ), which is poorly expressed in normal intestinal epithelium, but could be induced in inflamed and injured mucosa. The goal of this his study was to investigate the roles of P‐cadherin in regulating intestinal inflammation, mucosal repair and CAC.

P‐cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. Roles of P‐cadherin in intestinal inflammation and tumorigenesis in vivo were investigated using a mouse strain with total P‐cadherin knockout. Dextran sulfate sodium (DSS)‐induced colitis was utilized to study mucosal inflammation, whereas CAC was established using a classical azoxymetane (AOM)/DSS model. Severity of acute DSS colitis was not affected by P‐cadherin knockout, however, P‐cadherin null mice exhibited faster recovery after DSS withdrawal indicating accelerated repair of injured mucosa. No significant differences in the number and size of colonic tumors was observed in P‐cadherin null and control mice after AOM/DSS induced CAC. Consistently, CRISPR/Cas9‐mediated knockout of P‐cadherin in SK‐CO15 and HCA7 human colonic epithelial cell lines accelerated epithelial wound healing without affecting cell proliferation. The faster migration of P‐cadherin depleted cells was associated with diminished cell‐matrix adhesions and increased cell spreading. Loss of P‐cadherin resulted in activation of Src kinase and the pro‐migratory phenotype of P‐cadherin depleted cell was reversed by pharmacological inhibition of Src. Furthermore, accelerated epithelial wound healing caused by loss of P‐cadherin was prevented by Rac1 inhibition. Our findings highlight P‐cadherin as a negative regulator of intestinal epithelial would heling in vitro and mucosal repair in vivo. By contrast, this AJ protein appears to be dispensable for intestinal epithelial cell proliferation and CAC development.