bluuebunny/crossref_metadata_2025 huggingface dataset:
Tracing the Route of the Dorsal Nerve of the Clitoris
Authors: Gordon Victoria , Rowe Joanna , Grubb Lauren , Segars Larry , Surek Chris C. , McCumber Travis , Willett Gilbert , Olinger Anthony
Published: April 2020

OBJECTIVE

Literature indicates an increasing number of complications following the labiaplasty procedure which is a surgery with a known problem of underreported and understudied long‐term outcomes and complications such as loss of sensation in the female pudendum. The aim of this study was to determine the course and any variations of the dorsal nerve of the clitoris (DNC) after it pierces the perineal membrane to generate a surgical safe zone for procedures involving the female pudendum.

METHODS

Cadavers from the University of Nebraska Medical Center, Creighton University, and Kansas City University anatomy labs were dissected for this study. A shallow vertical incision was made from the pudendal cleft superiorly through the mons pubis. The glans clitoris was identified and used as a landmark while the fascia surrounding the body of the clitoris was carefully removed. The DNC was located where it pierces the perineal membrane and traced distally. The following measurements were taken: 1. from the point where the DNC pierces the perineal membrane to the urethra, 2. from the point where the DNC pierces the perineal membrane to the pubic bone, 3. from the angle of the clitoris to where the nerve branches on the dorsum of the body of the clitoris and 4. from that branch point to the distal most portion of the glans clitoris. Any anomalous branching patterns were noted.

RESULTS

Specific branching patterns were identified, mapped out and characterized into individual types. In addition to the patterns, the measurements described above were used to generate a surgical safe zone which can be used to avoid injury to the DNC during procedures involving the female pudendum. The variance was also calculated for each point of measurement to describe how much variation there is within a specific measurement to identify where the most caution is needed during surgery. Furthermore, it was determined if there were statistically significant differences between the right and left sides with regards to the nerve.

CONCLUSIONS

The findings of this study have allowed the investigators to map out a surgical safe zone for procedures involving the female pudendum to avoid injury to the DNC. Furthermore, multiple anomalous branching patterns were identified and the frequency of those patterns were reported. This information should be useful to physicians performing procedures near the DNC. Some of the specimens displayed anomalous branching patterns which did not fit any of the other types identified and physicians performing procedures should be aware of those patterns as well.

Support or Funding Information

There is no support or funding information.
Effect of the Novel Compound SMTP‐27 in a Mouse Model of Diabetic Kidney Disease
Authors: Shibata Keita , Yasuda Erika , Nishinaga Tomoka , Karasawa Sarara , Yamaguchi Tomoaki , Hashimoto Terumasa , Hasumi Keiji , Nobe Koji
Published: April 2020

Objective

Diabetic kidney disease (DKD) is the most common complication of diabetes mellitus and one of the primary causes of end‐stage renal disease in many countries. Although several candidate substances have shown efficacy against DKD, no effective clinical treatment methods have been established for preventing the progression of DKD. It was recently noted that the occurrence of oxidative stress, one of the contributors to the pathogenesis of DKD, is closely related to inflammatory cell mobilization, a process that increases inflammation by inducing the production of cytokines such as interleukin‐1β and tumor necrosis factor‐α. The present study aimed to evaluate the effect of Stachybotrys microspora triprenyl phenol‐27 (SMTP‐27), which is reported to have an anti‐inflammatory effect, in a mouse model of DKD.

Methods

The right kidneys of male type 2 diabetic db/db mice (6 weeks) were removed through small flank incisions under isoflurane anesthesia to induce DKD. Male C57BL/6J mice of the same age, subjected to the same surgery, were used as the control group. DKD mice are randomly assigned to the vehicle‐treated group, SMTP‐27‐treated group (30 mg/kg), or metformin‐treated group (300 mg/kg). They were treated from six to sixteen weeks of age: SMTP‐27 injections were administered intraperitoneally once every two days, and metformin was orally administered daily. Urinary albumin (Ualb), serum creatinine (Scr), and creatinine clearance (Ccr) were measured to evaluate renal function. In addition, histological examination of an excised kidney was performed by hematoxylin‐eosin staining and periodic acid–Schiff staining to evaluate tubular regeneration and glomerular sclerosis.

Results

Ualb and Scr levels were significantly higher and Ccr levels were significantly lower in type 2 diabetic db/db mice than in C57BL/6J mice. Moreover, tubular regeneration and glomerular sclerosis were significantly higher in db/db mice than in C57BL/6J mice. In the type 2 diabetes model, metformin only improved Ualb with causing antihyperglycemic effects; in contrast, SMTP‐27 significantly improved Ualb, Scr, and Ccr without inducing antihyperglycemic effects. In addition, SMTP‐27 reduced tubular regeneration, whereas metformin did not. Neither drug reduced glomerular sclerosis.

Conclusion

Our results showed that SMTP‐27 improved DKD without inducing antihyperglycemic effects. Thus, SMTP‐27 is a potential novel therapeutic agent for the treatment of DKD. In future studies, we plan to investigate the mechanism of action of SMTP‐27 against DKD.

Support or Funding Information

This study was supported in part by the Japan Society for the Promotion of Science KAKENHI (Grant Number 18K14958, awarded to KS; Grant Number 26460346, awarded to KN). SMTP‐27 was generously donated by TMS Co., Ltd. (Tokyo, Japan).
Correlation of Bone Density in Semi‐Aquatic and Aquatic Animals with Ecological and Dietary Specializations
Authors: Crowell Madelyn Galimore , Rahmat Sulman , Koretsky Irina
Published: April 2020

A successful transition from a terrestrial to an aquatic environment requires the acquisition of unique adaptations that fit the distinct habitat. Pachyosteosclerosis is a combination of pachyostosis (thickening of cortical bone) and osteosclerosis (thickening of medullary bone). This accumulation of bone increases the density and weight of the animals that exhibit these characters. Pachyosteosclerosis is commonly found in aquatic and semiaquatic animals such as sirenians (sea cows), odobenids (walruses), otariids (sea lions), phocids (true seals) and cetaceans (whales). Increased bone density occurred early in the transition from a terrestrial to a marine environment, present before many of the other morphological characters such as flukes, flippers and fins and behavioral characters such as oscillatory swimming associated with aquatic life appear. Pachyosteosclerosis helps to counteract buoyancy, allowing taxa to remain submerged longer in order to feed while expending little energy. This is clearly demonstrated in sirenians, which are known to feed on sea grasses in shallow waters near the coastline. In most other aquatic mammals, such as modern cetaceans, pachyosteosclerosis is followed by an osteoporosis‐like bone lightening for increased swimming speeds that allows for the active hunting of fast‐moving prey. Pinnipeds live in a wide range of habitats (from cold to warm waters, from marine to fresh water) and demonstrate varying feeding mechanisms, ranging from filter feeding to bottom feeding to catching fast prey. Increased bone density is seen in multiple species of seals in different geological ages from the Miocene (~ 23mya) to present. The morphological and ecological significance for seals with thicker bones is that they swim slower and hunt in shallower water, while seals with lighter bones swim faster and hunt fast moving prey. Bone microstructural disparity allows different species of seals to successfully live in sympatric habitats due to their ecological and dietary specializations. Bone density is one vital character that can be used to predict the specific ecological niche and feeding preference for pinnipeds. Some early hominids, such as Homo erectus, have been shown to have increased density in the bones also. These heavier, thicker bones would make it easier for early Homo to hunt in coastal waters for littoral food sources and would compensate for the lack of stability from bipedalism.

Support or Funding Information

Funding Provided by Torres Advanced Enterprise Solutions LLC and Just‐Julian Fellowship
Enhancing Strabismus Education through Interactive Learning Multimedia
Authors: Aziz Rem , Moss Heather , Claire Sheldon , Krebs Claudia
Published: April 2020

The objective of our project was to create an interactive online educational module that integrates biomechanics of the eye with its underlying anatomy to allow for a comprehensive understanding of pediatric strabismus and its clinical detection to prevent amblyopia.

Amblyopia is the leading cause of monocular vision loss in the pediatric population, affecting 3–5% of children. Untreated, it leads to irreversible visual impairment in an otherwise structurally normal eye and can have significant effects on quality of life that impact self‐esteem and restrict career opportunities due to loss of stereopsis. Majority of cases are caused by strabismus, a condition resulting in the ocular misalignment of the eye. Early intervention significantly improves vision outcomes and psychosocial well‐being of the patient, however requires prompt and skilled detection. The pediatric eye examination can be inherently challenging, particularly considering the low emphasis on ophthalmology teaching in medical school curricula. Primary care residents and undergraduate medical student trainees often report low levels of comfort performing screening eye examinations and managing common pediatric ophthalmological presentations. Integration of open educational resources such as interactive clinical skills modules can effectively advance ophthalmology education and promote prompt detection of pediatric strabismus, with the goal of mitigating serious ophthalmic complications and improving overall patient care.

To effectively address these educational challenges, we have developed an innovative online module that correlates ocular anatomy to biomechanical mechanisms, and provides practical guidelines to clinical examination. The module focuses on the pediatric eye examination, evaluating for amblyopia & strabismus, and was created through an international collaboration with experts in anatomy, education, and neuro‐ophthalmology to optimize content delivery and integrate clinical relevance. It offers a variety of interactive activities to engage student learning and concludes with a set of virtual cases entailing simulated clinical examination to assess acquired knowledge. Critical feedback will be collected using a student experience survey at the beginning and end of the module to solicit response regarding effectiveness of content delivery, preference of educational media, and comfort conducting a strabismus clinical examination.

This international effort aims to significantly improve confidence performing clinical examination, guide prompt amblyopia detection in the pediatric population, and prevent development of its irrevocable complications.
A Comparative Approach to Human Anatomy Outreach
Authors: Reed James A.
Published: April 2020

Bringing high school students to the anatomy lab allows them to reinforce topics introduced in their classroom by providing an opportunity for experiential learning. They are able to engage with human tissue, as opposed to models and textbooks. However, whereas often medical and undergraduate students have the luxury of a slower exposure to the dissection of human materials, visiting high school students must obtain a level of comfort very quickly. In an attempt to facilitate this, an outreach activity geared toward high school students visiting the Sanborn Anatomy Lab at the Geisel School of Medicine was developed utilizing a recently created collection of comparative, non‐human, anatomical material; which consists primarily of bones, brains, and hearts. Visiting students view and discuss this material prior to a similar activity using a human cadaver. It has been observed that students participating in this activity are more comfortable and remain engaged throughout the length of the visit. Pre and post surveys are being used to formally confirm observed behavior. It is also possible that this activity could better highlight anatomical concepts such as analogous vs homologous structures, body organization, and form and function than human tissue alone. While the goal of this activity is to make visiting students more comfortable when viewing human cadavers, it could also increase the scope of the lab to include outreach groups with primary interests in biomechanics, evolution, and zoology. Additionally, the small initial investment of creating this collection has increased the utilization and visibility of the Sanborn Lab throughout the Dartmouth College community, with courses from the departments of Anthropology, Biology, and Psychology and Brain Sciences currently utilizing the comparative material and lab. The creation and implementation of a comparative collection could easily be scaled and implemented at any facility conducting similar outreach with minimal financial investment.
SPARC: Parameters of Electrical Excitation and Block of the Rat Vagus Nerve
Authors: Pelot Nicole A. , Grill Warren M.
Published: April 2020

There is growing interest in bioelectronic medicines, where diseases are treated through electrical stimulation and block of the peripheral autonomic nervous system. Given the widespread connections of the vagus nerve from the brainstem to most truncal organs, applications include treating epilepsy, depression, obesity, heart failure, and rheumatoid arthritis. However, designing and programming the parameters of bioelectronic medicines require understanding the biophysical responses of small myelinated and unmyelinated autonomic fibers. Using in vivo electrophysiology, we quantified strength‐duration properties, activity‐dependent slowing (ADS), and responses to kilohertz frequency (KHF) signals for rapidly conducting (>2 m/s) and slowly conducting (<2 m/s) fibers in the rat vagus nerve.

We stimulated the cervical vagus nerve and recorded compound action potential (CAP) input‐output curves from the abdominal vagus nerve for pulse widths from 20 to 1000 μs. Thresholds were largest for the slowest fibres (0.5 to 1 m/s), especially at shorter pulse widths. Fits to the data using standard strength‐duration equations were qualitatively similar, but the estimates of chronaxie and rheobase varied substantially.

ADS describes the slowed conduction in peripheral axons resulting from persistent low frequency activity. Using a novel cross‐correlation CAP‐based analysis method, we measured ADS of ~2.3% after 3 min of 2 Hz stimulation, approximately constant across fiber conduction speeds. This is comparable to that reported for sympathetic efferents in somatic nerves, but much smaller than ADS in cutaneous nociceptors. We found greater ADS with higher stimulation frequency and non‐monotonic changes in conduction speed in select cases.

KHF signals can block neural conduction in peripheral axons, and we used CAP recordings to quantify the effects of KHF signals from 10 to 80 kHz on small autonomic fibers. Block thresholds were higher for more slowly conducting fibers, and block thresholds increased monotonically with frequency, in contrast to published findings indicating that block thresholds of unmyelinated axons vary non‐monotonically with frequency. Further, there are varied reports on the time for recovery of neural conduction after KHF block; we found that the carryover effect could last tens of seconds following 25 s of KHF signal.

The quantification of mammalian autonomic nerve responses to conventional and kilohertz frequency signals provides essential information for development of bioelectronic medical devices and for understanding mechanisms of action.

Support or Funding Information

This work was supported by Fulbright Canada (15122811), the Natural Sciences and Engineering Research Council of Canada (PGS M‐425353‐2012 and PGS D3‐437918‐2013), and Duke University (University Scholars Program, James B. Duke Fellowship, and Pratt School of Engineering Faculty Discretionary Fund).
Ageing signalling pathways: Quantifying the effects of protein interactions
Authors: Sadria Mehrshad , Layton Anita
Published: April 2020

Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of environmental changes. In mammalian tissues a deficit of these mechanisms is commonly associated with cellular aging and degenerative diseases related to aging, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as being able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. In particular, mTOR, AMPK, and sirtuins are known to play an essential role in the management of metabolic stress and in the mechanisms and metabolism of ageing of mammals. Given that the ageing process remains to be completely understood, the goal of this study is to gain insights into the interactions of the proteins in the ageing‐related signalling pathways, and how those interactions may impact longevity and related diseases. To achieve that goal, we have developed a computational model of ageing signalling pathways. Specifically, the insulin growth factor/mTOR pathway and the sirtuin pathway are represented. The model simulates the interactions among Akt, mTORC1, AMPK, NAD, and SIRT, and predicts their dynamics. The model can be used as an essential component to simulate gene manipulation, therapies (e.g., resveratrol and curcumin), calorie restrictions, and chronic stress, and assess their functional implications on longevity and ageing‐related diseases.

Support or Funding Information

This research was supported in part by the Canada 150 Research Chair program.
Muscle Stem Cell Depletion Blunts Skeletal Muscle Adaptation to Weighted Wheel Running
Authors: Dupont Alec Marc , Englund Davis , McCarthy John , Peterson Charlotte
Published: April 2020

Satellite cells, the resident muscle stem cell, are not required for short term skeletal muscle hypertrophy in mice when subjected to mechanical overload via synergist ablation, however, long term growth is blunted. This study aimed to further investigate the effect of satellite cell depletion on muscle adaptation using Progressive Weighted Wheel Running (PoWeR). We hypothesized that satellite cell depletion would reduce the adaptive response of skeletal muscle to the training stimulus. The Pax7‐DTA mouse model was used to deplete satellite cell content in adult skeletal muscle. PoWeR training involved adding weight to the running wheel of the mice incrementally over the 8‐week training period. Hindlimb muscles were excised at the 8‐week time point, and the plantaris was immunohistochemically analyzed. PoWeR induced an oxidative shift in fiber type distribution and increased muscle fiber cross sectional area (CSA). However, this increase in fiber CSA was blunted in mice without satellite cells. To investigate the blunted growth in satellite cell‐depleted mice, the plantaris was analyzed for extracellular matrix content and capillarization. We found that neither extracellular matrix buildup nor capillary density differed between satellite cell replete and depleted groups following PoWeR. These findings suggest that loss of satellite cells reduces the capacity for hypertrophy in response to PoWeR, but this was not due to excessive extracellular matrix accumulation. Further, satellite cells were not required for a robust fiber type shift in response to PoWeR training.

Support or Funding Information

This work was funded by the American Physiological Society’s UGSRF program and a grant from the National Institute of Health to Dr. Charlotte Peterson and Dr. John McCarthy (AR060701).
Circulating microparticles induced neutrophils chemotaxis through FOXO3a and the inhibiting role of dexmedetomidine in cardiorenal syndromes
Authors: Ou Jing-song , Yuan Hao-Xiang , Chen Cai -Yun , Li Yu-Quan , Li Yan , Xu Kang-Qing , Ou Zhijun
Published: April 2020

Rationale

Cardiorenal injury, also called cardiorenal syndromes, are the common complications in cardiac surgery. We previous demonstrated that circulating microparticles from patients with valvular heart disease (vMPs) and cardiac surgery contained inflammatory components and inhibited endothelium‐dependent vasodilation. However, the effects of vMPs on cardiorenal syndromes are unclear. Neutrophils chemotaxis plays an important role in cardiorenal syndromes. In addition, dexmedetomidine (DEX) could reduce cardiorenal syndromes in cardiac surgery. However, the effect of DEX on vMPs and neutrophils chemotaxis was unknown.

Objective

To investigate the effects and mechanisms of vMPs on heart and kidneys and the effect of DEX on vMPs and neutrophils chemotaxis.

Methods and Results

Circulating MPs were isolated from health subjects (nMPs) and patients with VHD. vMPs stimulated the C‐X‐C motif chemokine 4(CXCL4) and C‐C motif chemokine 5(CCL5) released from cultured human umbilical vein endothelial cells (HUVECs) and induced neutrophils chemotaxis. Silencing CXCL4 or CCL5 inhibited vMPs‐induced neutrophils chemotaxis. vMPs increased the expression of forkhead box proteins 3a (FOXO3a) and decreased the phosphorylation of AKT and FOXO3a, resulting in the transport of the FOXO3a from cytoplasm to nucleus and the activation of FOXO3a signaling pathway. Silencing FOXO3a reduced the vMPs‐induced CXCL4 and CCL5 as well as neutrophils chemotaxis. vMPs enhanced the levels of CXCL4 and CCL5 in plasma, the neutrophils accumulation, but inhibited the phosphorylation of AKT and FOXO3a and increased FOXO3a, leading to the activation of FOXO3a signaling pathway in the heart and kidneys, which could be inhibited by pretreated with DEX in vivo. Pretreated with DEX also reduced vMPs‐induced CXCL4 and CCL5 and neutrophils chemotaxis in cultured HUVECs.

Conclusion

vMPs induced CXCL4‐CCL5 to stimulate neutrophils infiltration in the heart and kidneys, which could be inhibited by DEX via FOXO3a signaling pathway. Our findings present a novel mechanism by which vMPs induce cardiorenal injury and may provided a new basis for the application of DEX in reducing cardiorenal syndromes in cardiac surgery.

Support or Funding Information

This research was financially supported by the National Natural Science Foundation of China (Grants 81670392, 81600382, 81770241, 81830013, 81970363 and Distinguished Young Scholar Grant 81325001), International Cooperation project (2015DFA31070) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee (Grant 2015A030312009), the Changjiang Scholars Program from the Ministry of Education of China, the Sun Yat‐sen University Clinical Research 5010 Program, Program of National Key Clinical Specialties.
The Catalytic Features of IDH1 Mutations Can Drive Neomorphic Activity, Phenotype Severity, and Inhibitor Binding
Authors: Sohl Christal D. , Matteo Diego Avellaneda , Wells Grace , Luna Lucas , Upadhye Viraj , Quichocho Giovanni , Chambers Jennifer M. , Miller Wade , Scott David , Schiffer Jamie M.
Published: April 2020

Isocitrate dehydrogenase 1 and 2 (IDH1, IDH2) balance metabolite levels by catalyzing the reversible NADP+‐dependent oxidation of isocitrate to a‐ketoglutarate (αKG). Mutations in these enzymes drive many cancers such as gliomas, leukemias, and chondrosarcomas by ablating the normal reaction and, critically, by catalyzing a neomorphic reaction: the NADPH‐dependent reduction of αKG to D‐2‐hydroxyglutarate (D2HG), an oncometabolite. Most mutations affect residue R132, and the structural and chemical diversity of the mutants observed in patients, including R132H/C/G/L/S/Q, is striking. The most common mutants in IDH1 and IDH2 serve as bona fide therapeutic targets, with selective mutant IDH inhibitors currently in the clinic. Using steady‐state kinetics methods, we recently found that the catalytic efficiency of both the normal and neomorphic reactions vary widely depending on the mutation at residue 132. Importantly, we have identified one mutant, R132Q IDH1, that preserves some wild type activity, catalyzes very highly efficient D2HG production both in biochemical and cellular models, and is resistant to allosteric mutant IDH1 inhibitors. We also show intriguing evidence that the catalytic efficiency of D2HG production appears to play a role in dictating the intensity of cellular phenotypes, allowing us to relate catalytic profiles to cancer phenotype severity. Finally, using molecular dynamics simulations and structural methods, we identified an interesting pathway of water molecules that may help drive inhibitor selectivity in mutant IDH1. By establishing the molecular mechanisms of tumor‐driving IDH mutant catalysis and inhibition, we can generate tools for predicting patient prognosis and inhibitor response, and ultimately identify new targets and handles for drug design.

Support or Funding Information

This work was funded by a Research Scholar Grant, RSG‐19‐075‐01‐TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).
Extracellular interaction between Neurotrophic factor‐α1 and HTR1E serotonin receptor promotes cell survival
Authors: Sharma Vinay Kumar , Xuyu Yang , Sanchez Daniel-Saiz , Kim Soo-Kyung , Goddard William A. , Loh Y. Peng
Published: April 2020

Neurotrophic factor α1 (NFα1), classically known as carboxypeptidase E (CPE) has a non‐enzymatic role in the survival of neurons and cancer cells via binding to a putative receptor. We have identified 5‐Hydroxy tryptamine receptor 1E (HTR1E), a serotonin receptor with unknown function, as a binding partner that interacts with NFα1/CPE extracellularly to mediate cell survival. Co‐immunoprecipitation using a human LN18 cells and pull‐down assay with NFα1/CPE and HTR1E expressed in HEK293 cells confirmed interaction of these two molecules. 125I NFα1/CPE binding studies demonstrated saturable, high affinity binding to HTR1E. Molecular docking studies revealed the surface interaction between NFa1/CPE and HTR1E via 3 salt bridges, further stabilized by hydrogen bonding. We also found that HTR1E was able to activate ERK/CREB signaling upon treatment with NFα1/CPE. Pretreatment of HTR1E stable cells with NFα1/CPE followed by H2O2‐induced oxidative stress prevented a decrease in pro‐survival protein BCL2 and reduced cytotoxicity. SiRNA knock‐down of HTR1E in U118 glioblastoma cells resulted in significant inhibition of these cancer cells survival. Immunocytochemical analysis of human hippocampus indicated co‐expression of HTR1E with NFα1/CPE in CA1‐3 and dentate gyrus, and cellular co‐localization at the cell membrane in CA3 neurons. This study has uncovered a novel role for HTR1E as a binding partner for NFα1/CPE, and their interaction activates the ERK‐CREB‐BCL2 pathway to promote cell survival.

Support or Funding Information

Division of intramural research, NICHD, NIH, Bethesda, MD, USA
Chronic kidney disease‐induced insulin resistance is mediated by increased phosphorylation of SGK1 and enhanced SGLT2 expression in NTS
Authors: Chen Hsin-Hung , Cheng Pei-Wen , Tseng Ching-Jiunn
Published: April 2020

Background

The serum and glucocorticoid inducible‐kinase‐1 (SGK1) expression is regulated during discrete developmental stages and pathological conditions, such as hypertension and diabetic neuropathy. In the present study, chronic kidney disease (CKD) in rats enhanced SGK1 function and regulation within the nucleus tractus solitarii (NTS). This study aimed to assess the regulatory mechanism of SGK1 in the NTS in rats presenting CKD‐induced insulin resistance.

Methods

Eight‐week‐ and nine‐week‐old male Wistar–Kyoto (WKY) rats underwent nephrectomy of the left and right kidneys, respectively. After subjecting the rats to CKD‐induced hypertension for 2 weeks, they were treated with SGK1 inhibitor (GSK650394, 100 mM) for another 2 weeks. Furthermore, the rats underwent 5/6 nephrectomy for 4 weeks; thereafter, they were tested for blood pressure, blood and urine chemistry, renal function, and protein expression. Mann–Whitney U test was used to compare the mean rank differences in scales of the general parameters between the CKD (sham, CKD, and CKD + GSK650394) groups, and the values were presented as mean ± SEM.

Results

Rats with CKD revealed proteinuria, lower urine sodium and chloride levels and hyperglycemia, while treatment with SGK1 inhibitor significantly decreased these effects. Immunoblotting analysis further revealed that phospho‐SGK1 (Ser78), phosphoinositide‐dependent kinase‐1 (PDK1), and sodium‐glucose co‐transporter 2 (SGLT2) expressions increased in the NTS of CKD rats. Furthermore, inhibition of phospho‐SGK1 (Ser78) by GSK650394 significantly decreased the expression of PDK1 and SGLT2 in the NTS of CKD rats.

Conclusion

Our results suggest that inhibition of SGK1 expression in the NTS by intracerebroventricularly injected SGK1 inhibitor decreases the fasting serum glucose by negatively regulating CKD‐induced SGK1 signaling, reducing SGK1 (Ser78) phosphorylation, and by inhibiting the PDK1 and SGLT2 expressions in the NTS. These results indicate that SGK1 regulates the cardiometabolic syndrome in the NTS of CKD rats.

Support or Funding Information

107‐2320‐B‐075B‐002‐MY2、107‐2320‐B‐075B‐001
Sensitizing Aptamer INSR‐A43 Enhances Insulin Sensitivity and Sensitizes Insulin Receptor Signaling Pathways
Authors: Park Mangeun , Yunn Na-OH , Ryu Sung Ho
Published: April 2020

Aptamers are single‐stranded oligonucleotides (DNA or RNA) and form a unique three‐dimensional (3D) structure based on its sequence. These aptamers have high affinity and specificity to target molecule and recognize 3D structure of target molecule surface. For this reason, aptamers have been in the spotlight as a universal capture. Especially, aptamers can modulate target protein as allosteric agonist or allosteric antagonist. However, aptamer‐based allosteric enhancer or sensitizer (hereafter ‐ sensitizer) has not been elucidated. Here, for the first time, we show the INSR‐A43, which is aptamer‐based sensitizer for the insulin receptor. Because insulin receptor plays a key role in regulation of glucose metabolism, it is important to specifically modulate the insulin receptor. INSR‐A43 binds to allosteric site of the insulin receptor and then increases insulin binding to the insulin receptor. Taken together, INSR‐A43 effectively enhances insulin sensitivity to the insulin receptor and sensitizes insulin receptor and insulin receptor‐related downstream signaling pathways. We expect that aptamers can be applied as an attractive tool for receptor sensitization.

Support or Funding Information

This research was supported by Grobal Research Laboratory (GRL) Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF‐2016K1A1A2912722)
Parental obesity exacerbates cardiovascular responses to acute stress in male, but not female, offspring
Authors: do Carmo Jussara M. , Moak Sydney P. , Dai Xuemei , Hall John E. , Silva Alexandre A. D. A.
Published: April 2020

The proportion of women and men of reproductive age who are overweight and obese has been increasing considerably, and maternal obesity and excessive gestational weight gain may predispose their offspring to health‐related consequences during childhood and adulthood. Obesity may also be associated with increased cardiovascular responses to mental stress. However, there is no available evidence on the effects of intergenerational obesity on cardiovascular responses to acute stress in adult offspring. In this study, we examined whether the pressor response to acute stress is exacerbated in obese offspring from obese parents. Offspring fed normal diet (ND) born from ND parents (N‐F1‐N, n=5 per sex) used as control, offspring fed high fat diet (HFD) born from ND parents (N‐F1‐H, n=5 per sex) and offspring fed HFD born from HFD parents (H‐F1‐H, n=5 per sex) were used. Parents were fed HFD or ND after they were weaned, while the offspring were placed on HFD from weaning until the end of experiments. Mice were implanted with telemetry probes for measurement of mean arterial pressure (MAP) and heart rate (HR). After a 10 day recovery period, baseline MAP and HR were continuously measured for 30 minutes before, during 5‐minute air‐jet stress consisting of pulses of compressed air directed at the animal’s forehead, and 30 minutes after the stress test. Male and female H‐F1‐H mice were heavier than N‐F1‐H and N‐F1‐N control mice (47.3±0.8 vs. 42.5±1.9 and 33.1±0.2 g and 41.1±0.1 vs. 37.2±0.1 and 24.1±0.1 g, respectively). Male H‐F1‐H mice had higher MAP (110±4 mmHg) compared to N‐F1‐H and control mice (100±6 and 99±3 mmHg). Acute air‐jet stress significantly increased MAP by 36±2 mmHg in male H‐F1‐H mice compared to only 26±3 and 28±3 mmHg in male N‐F1‐H and control mice, respectively. MAP remained significantly higher post stress in H‐F1‐H mice (133±2 vs. 114±6 and 110±3 mmHg). Baseline HR and HR responses to air‐jet were similar among the male groups. In females, however, baseline MAP and HR as well as MAP and HR responses to air‐jet stress test were similar among groups. These results indicate that male, but not female, offspring fed a HFD from obese parents have higher baseline blood pressure (BP) and greater BP responses during acute stress, and their BP remains elevated for a longer period post stress. These observations also suggest that parental obesity may have greater effect on male offspring cardiovascular system compared to female offspring.

Support or Funding Information

NIDDK 1RO1121411, NHLBI‐PO1HL51971, NIGMS P20GM104357 and U54GM115428
N‐ and C‐Terminal Alzheimer’s Aβ Heterogeneity Modulates the Balance Between Brain Clearance and Amyloid Formation
Authors: Ghiso Jorge , Cabrera Erwin , Neubert Thomas A. , Rostagno Agueda
Published: April 2020

Impaired brain clearance and abnormal Aβ degradation are today considered key events for the formation and progressive accumulation of soluble neurotoxic oligomers and the development of synaptic pathology, one of the strongest correlates to cognitive impairment in Alzheimer’s disease (AD). Clearance studies, mostly centered in monomeric Aβ40, have provided a basic assessment of the participating mechanisms, but the complex molecular and structural heterogeneity of the brain Aβ peptidome –going far beyond the classic Aβ40/Aβ42 dichotomy–has been largely overlooked. The most common heterogeneity resides in the multiple truncated fragments that consistently populate the Aβ peptidome and of which little is known in regard to their homeostatic regulation and potential contribution to disease pathogenesis. Tissue specimens from AD cases and transgenic models subjected to differential solubility extractions followed by immunoprecipitation coupled to mass spectrometry revealed the extent and characteristics of the multiple N‐ and C‐terminal truncated Aβ species. The data indicate that C‐terminal truncations increase Aβ solubility and abrogate oligomerization and fibrillogenesis, features clearly associated with clearance mechanisms, whereas N‐terminal truncations, particularly abundant components of parenchymal and vascular amyloid deposits in AD, non‐human primates, and multiple APP transgenic models drive enhanced pro‐amyloidogenic properties. Biophysical studies using synthetic homologues confirmed the differences in solubility further illustrating the contrasting oligomerization/fibrillization characteristics of the various Aβ truncated derivatives, and novel antibodies recognizing specific N‐ and C‐terminal truncations differentially immunolabeled amyloid deposits in AD brains and transgenic models. Intracerebral injections of monomeric and oligomeric radiolabeled homologues revealed striking differences in their brain clearance characteristics, showing that while soluble derivatives exhibited a fast brain removal, oligomerization largely increased brain retention, a characteristic particularly evident in fragments truncated at Phe4, topographically abundant in fibrillar, thioflavin positive plaque cores. Overall, our data indicate that degradation at the C‐terminus generates highly soluble Aβ fragments that are common components of interstitial and cerebrospinal fluids, and likely associated to catabolic/clearance mechanisms whereas truncations at the N‐terminus favor oligomerization and brain retention, with the potential to exacerbate the process of amyloid formation and self‐perpetuate the amyloidogenic loop.

Support or Funding Information

NIH grants AG059595 and AG051266, and the BrightFocus Foundation grant A2015275S
A High‐throughput CRISPR Interference Screen to Dissect Functional Regulators of GPCR/cAMP Signaling
Authors: Semesta Khairunnisa Mentari , Tian Ruilin , Kampmann Martin , von Zastrow Mark , Tsvetanova Nikoleta G.
Published: April 2020

The beta‐adrenergic (β2‐AR) receptor is a prototypical GPCR that controls important functions in the nervous, pulmonary, and cardiovascular systems. β2‐AR signaling involves multiple factors in both G‐protein dependent and independent signaling, resulting in diverse signaling outcomes. To understand the complexity of this pathway, we have performed the first unbiased, high‐throughput functional genomics screen of β2‐AR signaling using cAMP reporter and CRISPR interference (CRISPRi) silencing. We generated a reporter cell line using a construct encoding cAMP‐response element (CREB) fused to a ProteoTuner destabilizing domain and green fluorescent protein and another construct encoding catalytically‐dead Cas9 (dCas9) fused to the transcriptional repressor KRAB. Reporter cells were transduced with genome‐wide lentiviral sgRNA library to silence individual genes. After induction of β2‐AR signaling, we sorted the edited cells based on reporter expression and subjected them to deep sequencing and statistical analysis of sgRNA enrichment. We obtained 32 positive and 14 negative β2‐AR/cAMP signaling modulators enriched in factors involved in transcription initiation, RNA splicing, and RNA metabolic processes. We validated TP53 and NUP93 as novel positive modulators of β2‐AR signaling using dual luciferase cAMP assay and RT‐qPCR of cAMP‐dependent target gene, consistent with our cAMP reporter result. We found that the TP53‐dependent modulation of this pathway can partially be explained by lower transcription and surface expression of the β2‐AR receptors. These findings demonstrate a successful application of our reporter in a high‐throughput approach to identify previously uncharacterized modulators of the β2‐AR/cAMP pathway. This study reveals broader potential applications of our cAMP reporter as a platform to understand other aspects of GPCR signal transduction such as biased signaling, receptor‐specific molecular mechanisms, and drug discovery.

Support or Funding Information

This work is supported by the National Institute of Mental Health R00 award (MH109633) awarded to Nikoleta Tsvetanova.
A Role for Notch Signaling in Lymphoid Progenitor Development in the Bone Marrow
Authors: Sottoriva Kilian , Shao Lijian , Pajcini Kostandin
Published: April 2020

A major clinical obstacle after treatment of blood disorders with hematopoietic stem cell transplantation (HSCT) is the resultant long‐term impairment in T cell mediated adaptive immunity. Though T cell development in the thymus has been extensively characterized, there are significant gaps in our understanding of pre‐thymic T cell commitment. The Notch pathway is vital for the development of T cell progenitors in the thymus. We have confirmed a role for Notch signaling in the bone marrow Common Lymphoid Progenitor (CLP) population which suggests that Notch signals may instruct lymphoid progenitors towards the T cell fate even before exiting the bone marrow. Using a genetic Notch1 hypomorhic mouse model, we find defective T cell lineage development as early as the CLP, with no defect in B cell or NK cell development downstream of the CLP. We further generated a list of genes dependent on Notch signaling within the CLP population using RNA sequencing. One downstream target of Notch in CLPs is IL21r, which we have confirmed plays a role in the proliferation of Notch activated CLP cells in the bone marrow. Our work uncovers a T cell lineage developmental pathway initiated by Notch signaling in the bone marrow CLP population.
Pleiotropic Activation of Endothelial Function by Angiotensin II Receptor Blockers is Crucial to Their Protective Anti‐vascular Remodeling Effects
Authors: Tehrani Arash Y. , Fameli Nicola , White Zoe , Esfandiarei Mitra , van Breemen Casey , Bernatchez Pascal
Published: April 2020

Rationale

Endothelial function is strongly associated with vascular health and homeostasis. However, therapeutically relevant approaches capable of stimulating the protective properties of the endothelium remain elusive.

Objective

To determine whether modulation of the Angiotensin II (AngII) system with Angiotensin receptor blockers (ARBs) can improve endothelial function and promote downstream vascular homeostasis.

Methods and Results

We show that all 4 ARBs tested (losartan, telmisartan, olmesartan, and valsartan) can promote robust chronic activation of endothelial nitric oxide (NO) in mice. With telmisartan, a well‐known pleiotropic ARB, in vivo treatment lead to a 58% inhibition of vascular contractility after two weeks of treatment and up to 77% at 6 months, in a fully L‐NAME sensitive fashion. Endothelial NO synthase (eNOS) knockout (KO) mice showed complete resistance against the endothelial function effects of telmisartan, whereas the Angiotensin converting enzyme inhibitor (ACEi) captopril showed little to no endothelial function activity at a similar blood pressure (BP) lowering dose. In a myograph chamber, direct stimulation of aortic vessels with telmisartan in the absence of AngII stimulated endothelial function, confirming pleiotropism. Moreover, in vitro stimulation of cultured endothelial cells caused rapid intracellular Calcium ([Ca2+]i) mobilization, which is a known upstream event of eNOS activation. In vivo, telmisartan completely abolished aortic wall remodeling in aging animals as well as a well‐established mouse model of Marfan syndrome (MFS), which causes accelerated and pre‐mature vascular aging and remodeling of the aortic root. In addition, in vivo inhibition of NOS activity with L‐NAME rendered telmisartan inactive in a BP‐independent manner.

Conclusion

ARBs have unique and robust endothelial function activation properties independent of their inhibitory effects on the AngII system. Direct stimulation of the endothelial NO system with ARBs results in vascular protection and homeostasis independent of BP lowering effects, which supports a broader prophylactic use of this class of medication.

Support or Funding Information

Supported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.
Heart Rate Response of Chicken Embryos to Room Temperature at Different Ages
Authors: Sycip Oliver C. , Brundage Cord M.
Published: April 2020

Chicken (Gallus gallus domesticus) embryos are used as a research model. Embryotic heart rate (HR) can be monitored non‐invasively for health as well as a physiological response to experimental stimuli. References ranges for embryotic HR vary with cardiovascular and embryonic development. These may be influenced by the environment in which they are measured. A decline in heart rate (HR) has been documented in embryos following removal from incubation. This was hypothesized to be a response to the drop in ambient temperature. The magnitude and duration of this response may vary with embryonic development. To evaluate the effect of ambient temperature on chicken embryo HR, 10‐day, 15‐day, and 18‐day old chicken embryos (n = 20, 15 and 15 respectively) were placed on HR monitors for 10 min at both incubation (37.7 ± 0.1 °C) and room (25.6 ± 0.4 °C) temperatures. The pattern of HR change was similar in both temperature groups. HR predictably increased with embryonic age (p < 0.05); HR across all ages decreased similarly (p > 0.10). After a decrease in HR after 3 min (−30 ± 39 bpm, p < 0.01), HR did not continue to decline (p > 0.05). Differences in both HR between embryotic stages and changes in HR over the 10 minute trials were temperature independent (p > 0.05; p > 0.10). This would suggest that chicken embryo research can be conducted at room temperature within 10 min (in 10 – 18‐day embryos) without temperature significantly impacting HR. Initial decreases in HR may not be due to temperature, but rather other factors associated with movement and manipulation. When looking at changes in HR, egg handling procedures and embryo age should be considered and controlled.

Support or Funding Information

Research reported in this publication was supported by the MENTORES (Mentoring, Educating, Networking, and Thematic Opportunities for Research in Engineering and Science) project, funded by a Title V grant, Promoting Post‐baccalaureate Opportunities for Hispanic Americans (PPOHA) | U.S. Department of Education, Washington, D.C. PR/Award Number: P031M140025. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Education.
A Glial Cell Inhibitor Blocks Epithelial Barrier Repair in a Pig Model of Intestinal Ischemia
Authors: Sheridan Ana , Pridgen Tiffany , Odle Jack , Van Landeghem Laurianne , Blikslager Anthony , Ziegler Amanda
Published: April 2020

The gut epithelium functions as a barrier against toxic luminal contents which can repair efficiently after injury to prevent systemic illness and death. However, we have shown that repair is severely hindered in neonates as compared to juveniles in our translational pig model of intestinal ischemia. The subepithelial enteric glial cell (EGC) network is known to promote epithelial repair by paracrine signaling mechanisms. This EGC network develops postnatally; therefore, we believe this repair defect in neonates is due to an underdeveloped EGC network. In support of this, we hypothesize that EGC inhibition in juveniles will block epithelial barrier repair after ischemic injury mimicking the neonatal phenotype. Ischemia‐injured jejunum of juvenile pigs was recovered ex vivo with and without fluoroacetate (FA), a glial inhibitor. Transepithelial electrical resistance (TEER) was monitored as a measure of barrier function, and tissues were collected for imaging analysis and primary EGC culture. FA inhibited TEER recovery at all tested concentrations (P<0.0001). Histology showed 500μM FA optimally inhibits epithelial repair (P=0.0196) without directly damaging the epithelium (P=0.5509). Cellular metabolism marker c‐fos has been optimized for immunofluorescence in control tissues and ongoing imaging work will validate selective inhibition of EGC metabolism by 500μM FA. In addition, pig EGC isolation and culture has been optimized, so that future work will confirm inhibitory effects of FA on EGC signaling functions in vitro. Understanding the development of glial‐epithelial crosstalk in barrier repair will ultimately guide novel clinical solutions to improve outcomes in neonatal patients affected by intestinal injury.

Support or Funding Information

UNC CGIBD Large Animal Models Core (P30 DK034987), UNC CGIBD Basic Science Research Training Fellowship (NIH T32 5T32DK007737), UNC CGIBD Pilot Feasibility Grant (P30 DK034987), USDA National Institute of Food and Agriculture (Projects 1007263 and 07985)