bluuebunny/crossref_metadata_2025 huggingface dataset:
Chitosan Oligosaccharide Ameliorates Kidney Injury by Improving Intestinal Barrier Dysfunction and Lipid Metabolism in Obese‐insulin Resistant Rats
Authors: Sutthasupha Prempree , Promsan Sasivimon , Phengpol Nichakorn , Pichyangkura Rath , Muanprasat Chatchai , Lungkaphin Anusorn
Published: May 2022

Introduction

Obesity remains a global health problem. High‐fat diet (HFD) consumption increases the risk of type 2 diabetes and initiates kidney injury. Chitosan Oligosaccharide (COS) provides anti‐obesity effects through several mechanisms. However, the effects of COS on preventing obesity‐related kidney injury in obese‐insulin resistance have not been studied. A novel action of COS in activating intestinal tight junction assembly was reported in in vitro via calcium sensing receptor‐mediated AMP‐activated protein kinase (CaSR/AMPK) activation. HFD‐induced gut dysbiosis is related with intestinal barrier dysfunction and systemic low‐grade inflammation. Therefore, AMPK activation might be a therapeutic target for preventing obesity‐related complications. Here, we explored the protective effects of COS in obesity‐induced kidney dysfunction and the involved molecular mechanisms. We hypothesized that COS might exert renoprotection against obesity‐induced kidney injury through increasing intestinal barrier integrity and lipid excretion.

Methods

Male Wistar rats were fed with HFD for 16 weeks to induce obesity. Next, HFD rats were randomly divided into 4 groups: HFD, HFD treated with COS 5 or 10 mg/kg/day (COS5 or COS10) and HFD treated with metformin 30 mg/kg/day (MET). The interventions were orally treated for 8 weeks. At the end of experiment, blood, feces, intestinal and renal tissue samples were collected for further investigations.

Results

After 16 weeks of high‐fat diet feeding, obesity and glucose intolerance were presented in HFD compared to normal diet (ND) rats, as indicated by the significant increases in body weight (BW), plasma cholesterol and total area under the curve for glucose (TAUCg). Kidney injury were observed at week 24, as shown by elevated kidney injury score and serum creatinine. Treatment with COS5 or COS10 significantly decreased BW, hypercholesterolemia, glucose intolerance and kidney dysfunction. Interestingly, COS10 markedly increased fecal lipid excretion compared with HFD and MET. In addition, intestinal CaSR, p‐AMPK/AMPK and tight junction protein claudin‐1 expressions were downregulated in HFD which were upregulated by COS10 and MET. Renal TNFαR activation and increased proinflammatory cytokines were detected in HFD. Moreover, renal apoptosis markers, Bcl‐2‐associated X protein (Bax) and cleaved caspase‐3, were increased, along with the suppressing of anti‐apoptotic B‐cell lymphoma‐2 (Bcl‐2) in HFD. Treatment with COS10 or MET markedly alleviated renal inflammation and apoptosis.

Conclusion

COS exerted renoprotection through attenuating renal inflammation and apoptosis as a result of improving intestinal barrier via CaSR/AMPK activation and lipid absorption in obese rats. Therefore, COS might be an effective new dietary supplement for treating obesity and its related complications in clinical use.
Effects of pituitary hormones deficiency from the specific pituitary lobes on learning, memory and survival behavior in the Wistar rat
Authors: Quintanar‐Stephano Andrés , Limón‐Mendoza Adrian , López‐Rico Martín , Muñoz‐Tabares José N. , Ramírez‐Rojas Norma A.
Published: May 2022

The role of the different adenohypophyseal hormones (GH, PRL, ACTH, TSH, LH and FSH) and neurohypophyseal (AVP and OXY) hormones on animal behavior, memory and learning have been stablished, however the role of lobe‐specific hormones on these cerebral functions has been not completely dilucidated. Here we study the specific pituitary hormone deficiencies in hypophysectomized (HYPOX), with anterior lobectomized (AL) and neurointermediate lobectomized (NIL) rats, subject to novel object recognition (NOR), forced swim (FS) and prepulse inhibition (PPI) tests. For comparisons the intact control (IC) and sham operated (SHAM) groups were also included. The NOR test that evaluate different phases of learning and memory in rodents, showed that memory discrimination index (DI) significantly increased in the NIL group, indicating that AVP and OXY deficiency negatively affect the ability of short and medium‐term memory. The FS test assess survival and motivation behavior; results showed that AL, NIL and HYPOX groups induced a loss in motivation and will to survive (increased immobility time); these observations may be partially explained by: 1) the significant deficiency of metabolic hormones (GH, ACTH‐corticosterone, TSH‐thyroid hormones and LH‐FSH‐testosterone hormones) whose decrease also induced significant loss in both mass muscle and body weight in AL and HYPOX groups and, 2) the decreased survival and motivational behavior induced by the AVP and OXY deficiency (NIL‐induced), thus supporting the view of the motivational and survival role of these hormones. The IPP test evaluates the rat ability to respond to potentially harmful acoustic stimuli and long‐term memory; results showed that AL animals did not show any sign of learning (expressed as very low inhibition percentage), whereas, interestingly, the HYPOX group (without any pituitary hormones) the long‐term memory was not significantly affected. In conclusion these results support the important role of the several pituitary hormones mainly neurohypophyseal hormones (AVP and OXY) controlling important brain functions necessary for learning, memory, and survival. These experimental models will allow in the future to study specifically the role of each pituitary hormone on the different aspects of animal behavior such as memory, motivation, generosity, adaptation, among others.
Dissecting the language of physiology students
Authors: Petzold Andrew M. , Nichols Marcia D.
Published: May 2022

Scientists are often criticized as being poor communicators who are unable to reach broader audiences due to confusing verbiage, awkward grammar and generally poor writing. These afflictions often translate into an inability to translate their science to others ‐ which in turn can lead to a general skepticism of scientific dissemination. Unfortunately, these misgivings that derive from science communication issues can have wide‐sweeping, real‐world consequences ranging from misinterpretation of scientific findings, to misunderstanding regarding the scientific process to outright scientific misinformation. How to communicate science is an important skill that must be gained through a combination of practice and teaching. Since many aspects of science communication begin to be engrained during their undergraduate science courses, we wanted to ascertain the roots of these communication issues. We examined student’s use of language across two Anatomy and Physiology courses – one aimed at non‐majors and one aimed at health science majors. Specifically, we analyzed formal written submissions form both classes to see how students use the common scientific conventions of the passive voice and nominalization.
The E3 ubiquitin‐protein ligase Nedd4‐2 regulates the sodium chloride cotransporter (NCC) but is not required for a potassium‐induced reduction of NCC expression
Authors: Rosenbaek Lena , Petrillo Federica , Van Bemmelen Miguel , Staub Olivier , Murali Sathish , Fenton Robert
Published: May 2022

Study objective

The activity of the sodium chloride cotransporter (NCC) in the kidney distal convoluted is important for kidney sodium (Na+) and potassium (K+) handling. NCC abundance is reduced by high plasms K+ levels. The E3 ubiquitin ligase neural precursor cell expressed developmentally downregulated 4‐2 (Nedd4‐2) is involved in regulation of NCC, but its precise role is unclear. The aim of this study was to examine the functional role of Nedd4‐2 on NCC regulation and whether Nedd4‐2 is involved in the high K+ effects on NCC.

Hypothesis

Nedd4‐2 regulates NCC membrane abundance by influencing NCC trafficking or degradation and plays an important role in the high K+ induced reduction in NCC.

Methods

In vitro studies were performed using tetracycline inducible MDCKI cell lines stably expressing human NCC combined with Nedd4‐2 knockdown using shRNA. The cell lines were characterized using immunoprecipitation coupled to biotin‐based membrane abundance assays. The effects of normal or high extracellular K+ on NCC in ex vivo isolated mouse kidney tubules from wildtype or Nedd4‐2 knockout (KO) mice were assessed.

Results

In MDCK cells the levels of NCC and phosphorylated NCC (pNCC) were increased with Nedd4‐2 deletion, and NCC levels on the membrane were elevated. No significant changes were seen for the SPS1‐related proline/alanine‐rich serine‐threonine kinase (SPAK) and phosphorylated SPAK (pSPAK) after Nedd4‐2 knockdown. Deletion of Nedd4‐2 had no effect on the endocytosis rate of NCC, but NCC half‐life was increased. In tubules high K+ reduced total and pNCC regardless of genotype. The levels of the inwardly rectifying potassium channel subunits Kir4.1 and Kir5.1 were increased in Nedd4‐2 KO tubules. High K+ treatment decreased the level of Kir4.1, but not Kir5.1, only when Nedd4‐2 was eliminated. pSPAK was decreased with high K+ treatment regardless of the genotype.

Conclusion

Nedd4‐2 is involved in ubiquitylation of NCC, regulation of NCC membrane abundance and degradation, but it does not play role in the K+ induced inhibition of NCC.
Quantitative Analysis of HPV E2 Binding to DNA Binding Sites and Sequence Variants
Authors: Evande Roxanne , Yilmaz Gulden , Biswas Subhasis
Published: May 2022

Many proteins, such as transcription factors, bind to DNA sequences within their genome to initiate different biological processes. In the human papillomavirus (HPV), the E2 protein controls the initiation of DNA replication through interaction with its binding sites. The HPV genome contains four binding sites for the E2 protein in a conserved spatial arrangement that frequently includes a 12 bp consensus sequence, ACCG(N4) CGGT. We have previously studied the strength of this interaction using the electrophoretic mobility shift assay (EMSA). The results showed that a single nucleotide variation in the E2 binding site consensus sequence can play a role in viral DNA replication. To expand on these findings and quantitatively correlate the binding affinity of the sequence variants, we further analyzed these interactions by expanding the previous binding studies. This project improved the initial binding affinity assay by incorporating an optimized recombinant protein and fluorescent probes. The qualitative binding results remained the same and to quantitatively observe the interaction we will be using surface plasmon resonance (SPR) to observe the Kon and Koff rates. Due to its conserved nature, the HPV E2 protein has been deemed a potential drug/therapeutic target. Hence, quantitative analysis of this interaction is important for drug development and therapeutic research.
Internal Melanin and Oxidative Stress: An Investigation of Lizard Livers
Authors: Wittle Liora E. , Del Gaizo Moore Victoria
Published: May 2022

The habitats of two brush lizards that reside in Arizona, U. ornatus and U. graciosus, overlap and a hybrid species has arisen over time that has the nuclear DNA from one introgressed with the mitochondrial DNA from the other. Previous studies by Haenel and Del Gaizo Moore have shown that the mitochondria in these hybrid lizards are much more inefficient than those in the two parental species. It was observed that all three species of lizards have internal melanin in their livers, with the hybrids possessing much higher amounts. One hypothesis explaining the higher melanin content in the hybrid’s livers is that it helps protect their inefficient mitochondria from oxidative stress. In order to test this hypothesis, we sought to induce expression of melanin in liver cells using 1,2‐dimethyl‐3,3‐butanadiol (1,2DM‐3,3BD). Initially, human epithelial kidney (HEK) cells were treated with 12.5‐37.5 mM of 1,2DM‐3,3BD for 6 days and their capacity to resist oxidative stress was tested using a CellROX and cell viability flow cytometry assay. Melanin induction in HEK cells was small and therefore no protection was afforded against oxidative stress. Next, a protocol was developed to isolate primary liver cells from North Carolina tree lizards. Currently, different methods are being tested to maintain these primary cell cultures long enough to treat it with 1,2DM‐3,3BD to determine if melanin expression can be induced in the cells.
Endothelial (Dys)function in Women With Androgen Excess Polycystic Ovary Syndrome: Effects of Body Composition and Ethinyl Estradiol Supplementation
Authors: Berbrier Danielle , Leone Cheryl , Adler Tessa , Bender Jeffrey , Taylor Hugh , Stachenfeld Nina , Usselman Charlotte
Published: May 2022

Polycystic ovary syndrome (PCOS) has been associated with endothelial dysfunction. However, whether endothelial dysfunction occurs as a result of obesity in androgen excess PCOS (AE‐PCOS) remains unclear. The purpose of this study was to compare endothelial function between lean and overweight/obese (OW/OB) women with AE‐PCOS. We hypothesis that OW/OB women with AE‐PCOS would demonstrate impaired endothelial function relative to lean women with AE‐PCOS as well as both lean and OW/OB controls without any form of PCOS. We also tested the hypothesis that short‐term ethinyl estradiol (EE) supplementation improves endothelial function in both lean and OW/OB women with AE‐PCOS. We assessed conduit vessel endothelial function in 14 women with AE‐PCOS (lean: n=7; OW/OB: n=7) and 12 controls (CTRL; lean: n=6, OW/OB: n=6) using flow‐mediated dilation (FMD). FMD responses were assessed twice: before (baseline; BSL) and on the last day of 7‐day oral EE (post‐EE; 30mg/day) and quantified as the peak increase in diameter during reactive hyperemia (%FMD), shear rate, and low flow‐mediated vasoconstriction (LFMC). BSL %FMD was attenuated in lean AE‐PCOS relative to both lean CTRL (5.2 ± 1.5% vs10.3 ± 2.6%, P<0.01) and OW/OB AE‐PCOS (5.2 ± 1.5% vs6.6 ± 0.9%, P=0.04). A negative correlation between BSL %FMD and free testosterone was observed in lean AE‐PCOS only (R2=0.68, P=0.02). %FMD was unchanged from BSL to post‐EE in lean AE‐PCOS (5.17±1.5 vs5.17±1.1, P=0.99). Post‐EE, %FMD was lower in lean CTRL (10.3±2.6% vs7.6±1.2%, P=0.03), but increased in both OW/OB CTRL (7.3±0.1.2% vs 10.4±2.5%) and OW/OB women with AE‐PCOS (6.6±0.92% vs 9.6±1.7%; main effect of EE, P<0.01). Post‐EE %FMD changes from BSL were associated with EE‐induced changes in LFMC and shear rate. Our data indicate that lean women with AE‐PCOS exhibit more severe conduit vessel endothelial dysfunction than OW/OB women with AE‐PCOS. Our data also indicate that short‐term EE supplementation affects LFMC and shear rate (and therefore %FMD) differently across healthy lean women and OW/OB women with and without AE‐PCOS.
Novel Roles of FFAR4 in Macrophage Foam Cell Formation
Authors: Stuttgen Gage M. , Sahoo Daisy
Published: May 2022

Free fatty acid receptor 4 (FFAR4), also known as G‐protein coupled receptor 120 (GPR120), is a long‐chain unsaturated fatty acid receptor expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. While FFAR4 is best known for its role its role in preventing obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and cardiovascular disease (CVD). Given FFAR4’s importance in anti‐inflammatory signaling and high expression levels in macrophages, we designed experiments to test the hypothesis that FFAR4 prevents the development of atherosclerosis by reversing macrophage foam cell formation, a hallmark of early atherogenesis. In these studies, we isolated peritoneal macrophages from wild‐type C57/BL6J mice and incubated them with oxidized low‐density lipoprotein (oxLDL) to generate foam cells. We then investigated the effects of FFAR4 activation by GW9508 (a synthetic agonist) on lipid accumulation, cytokine secretion, and cholesterol efflux. Activation of FFAR4 by GW9508 decreased macrophage secretion of pro‐inflammatory cytokines. We also found that activation of FFAR4 with GW9508 reduced lipid accumulation in macrophages as observed by decreased Oil Red O staining and reduced cellular cholesterol content. Additionally, activation of FFAR4 by GW9508 increased [3H]‐cholesterol efflux to high‐density lipoprotein (HDL). Interestingly, the increased efflux was accompanied by decreased scavenger receptor CD36 expression (that mediates oxLDL uptake) and increased expression of ATP binding cassette transporters, ABCA1 and ABCG1 (that mediate cholesterol efflux). Taken together, our results support an exciting and novel role for FFAR4 in the reversal of foam cell formation and could emerge this receptor as a new target for treating CVD by preventing accumulation of atherosclerotic plaque.
Engaging Students Equitably in Biological Systems Modeling
Authors: Anjur Sowmya
Published: May 2022

It has been my goal as an educator to engage students in meaningful and innovative assessments that are hands‐on, engage their creativity and improve their interdisciplinary understanding. At the same time, the engagement need to be equitable as well, to recognize each student for their individuality and creativity. To this end, I created my Pathophysiology course with the objective of teaching students how to model biological systems. The course is a one semester course that is based on homeostasis maintenance in the body and how homeostatic disruptions result in symptoms of pathologies. Students are required to understand how homeostasis is maintained in individual organ systems and encouraged to model their understanding of homeostatic disruptions in ways other than biology, such as computer science and mathematics. This gives them an opportunity to use their creativity as well as apply their learning in other disciplines to create an interdisciplinary model of a biological system. Students have indicated that they appreciate being able to use their creative talents and non‐biological skills to model biological systems. Being able to use their non‐biological skills in a biology elective both encourages their problem solving skills and gives them confidence. Student learning is assessed through pre and post assessments and shows significant improvement in student understanding.

Promoting equity in assessments has been challenging. There is a need to recognize the individual skills and learning patterns of each student and allow for this in creating assessments. I decided to replace two written assessments with hands on projects such as the heart model project and the graphic novels project. My purpose was to minimize memorization and promote integrative learning in a fun way. The heart model project replaced the cardiovascular system unit test. A variety of materials are provided to students for making life sized heart models, and instructor approval of student blueprints is required before beginning construction. Extra credit is offered if the model “works”. A second assessment, the graphic novel project, replaced student presentations and were much preferred by the students for the opportunities offered to demonstrate their drawing and creative skills. Every student felt included (as indicated on their surveys) and enjoyed the interdisciplinary integration of modeling in the course, an exercise in logical thinking that is applicable to any other discipline as well.
Oral Microbiota of Amazonian Kichwa Infants From Equador During the First Six Months of Life
Authors: Maldonado Lino Diana , Diaz Byron , Vanden Berghe Wim , Cevallos‐Cevallos Juan M. , Orellana‐Manzano* Andrea
Published: May 2022

Background

For public health policy, this study aimed to identify the oral microbiota in babies of the Amazonian Kichwa (AK) population of Ecuador born vaginally during the first six months of life.

Method

The study was descriptive, observational, cross‐sectional, where the bacterial communities of infants were analyzed. Twenty‐five infants self‐identified as AK were recruited from 2 rural areas (Ñucanchi‐Llacta and Pano) and one urban area (center of Tena) of Ecuador. Saliva samples were collected from the infants using the self‐collection kit. DNA was extracted from salivary samples, bacterial amplicons (16S) were generated and sequenced using Nanopore MinION, and the sequences were pre‐processed and data analyzed using EPI2ME.

Results

High bacterial diversity was observed in infants' saliva [2191 operational taxonomic units with 1% divergence]. Firmicutes and Proteobacteria were the predominant bacterial phyla present in all samples. A total of 10 bacterial genera were present in our data set. The predominant genera in infant saliva were Streptococcus, Staphylococcus, Bacillus, Gemella, and Veillonella.

Conclusion

our data show a rich bacterial community in the baby's oral cavity. The oral microbiota of the infants was relatively stable during the period studied and was very similar between them. Such similarity may result from maternal child care habits (e.g., kissing on the mouth). Further research is required to characterize the development of the oral microbiota early in life and identify environmental factors that affect colonization and the risk of oral and gastrointestinal diseases.
Phospholipase D2 Loss Impairs Low Salt‐Induced Increases in Steroidogenic Gene Expression with No Effect on Serum Aldosterone Levels
Authors: Spaulding Shinjini C. , Choudhary Vivek , Kaddour‐Djebbar Ismail , Bollag Wendy B.
Published: May 2022

Primary aldosteronism (PA), in which plasma aldosterone levels are normal or elevated relative to suppressed plasma renin levels, is the most frequent cause of secondary hypertension. PA accounts for 5‐10% of hypertension cases and up to 20% in those with resistant hypertension. Aldosterone is a mineralocorticoid hormone responsible for maintaining fluid volume, electrolyte balance, and blood pressure homeostasis; it is tightly regulated by the renin‐angiotensin‐aldosterone system. Our laboratory has previously shown that phospholipase D2 (PLD2) mediates angiotensin II‐induced aldosterone synthesis and secretion in vitro. Therefore, we hypothesized that PLD2 loss would result in decreased aldosterone production in vivo. To test this hypothesis, 11‐week‐old male and female wild‐type and PLD2‐/‐ mice were fed a high salt (1% NaCl) or a low salt (0.02% NaCl; to raise endogenous serum angiotensin II levels) diet for 24 hours. Adrenal steroidogenic gene expression and serum aldosterone, corticosterone, and electrolytes were measured by qRT‐PCR, radioimmunoassay, ELISA, and atomic absorption spectrometry, respectively. The PLD2‐/‐ male mice exhibited increased StAR, Dab2, and CYP11B1 expression; further, PLD2 loss impaired low salt‐induced increases in StAR, Dab2, and CYP11B1 expression; however, PLD2 loss had no effect on CYP11B2 expression or serum aldosterone, Na+, K+, and corticosterone levels in these male mice. These data suggest that increased steroidogenic enzyme expression may compensate for PLD2 loss in order to maintain aldosterone secretion in the males. On the other hand, the PLD2‐/‐ female mice exhibited decreased StAR and CYP11B1 expression and serum Na+ levels as well as impaired low salt‐induced increases in StAR, CYP11B2, Dab2, and CYP11B1 expression. PLD2 loss had no effect on serum aldosterone, K+, or corticosterone levels in these female mice. It is possible that other compensatory mechanisms (not involving steroidogenic enzymes) might be involved in maintaining aldosterone secretion in the females. Together, our findings suggest a likely importance of PLD2 in mediating aldosterone production and steroidogenic enzyme gene expression, with differential effects in male and female mice.
Homologues Phospholipases A2 isolated from Naja naja atra snake venom exhibit different activity against intact human erythrocytes
Authors: Cabrera Hernández Luis O. , Boyne Mascarell Juan , Maldonado Hernández Rafael , Quesada Orestes
Published: May 2022

Phospholipases A2 (PLA2) catalyze the hydrolysis of fatty acid esters in the 2‐position of 3‐snphospholipids and are found in intracellular and secreted forms. Secretory PLA2s (sPLA2s) are small proteins (13‐15 kDa) classified into five groups based on primary structure differences. Despite their structural similarity, sPLA2s show a wide range of enzymatic and pharmacological activities. Most sPLA2s have high activity toward anionic membranes due to cationic residue presence on their binding recognition site (BRS). Within the same snake venom, several PLA2s can be found, which present differences in their ability to hydrolyze phospholipids in biological bilayers.

In this study, Chinese cobra, Naja naja atra (N. n. atra) venom was purified by Size Exclusion Chromatography using a Superdex 75 and cation exchange using a column packed with Carboxymethyl Cellulose (CM‐Cellulose) to pre‐separate protein components from the venom. Subsequently, fractions displaying PLA2 activity were pooled, concentrated, and applied to a GE mini‐CM‐Cellulose column coupled to the AKTA Fast Protein Liquid Chromatography (FPLC) system. The final purification presents four peaks but only two present PLA2 activity (Band D and E). Peak purity was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).

We have shown that hydrophobic residue presence on the PLA2 BRS is crucial for binding and hydrolytic activity toward human erythrocyte membranes. PLA2 activity was assayed against human erythrocytes and monomeric substrate to determine differences in hydrolytic activity. Primary sequence alignment shows 94% homology, however Band E presented 40% less activity against human erythrocytes, despite having 100% active site homology. Molecular modeling of both sPLA2s revealed differences at the Cys‐19 and Trp‐19 position and spatial orientation differences at the Trp‐61, Tyr‐63 Phe‐64, and Lys‐65 positions. Due to amino acid differences at the BRS, differences in hydrolytic activity may be linked to the anchorage of sPLA2 to the erythrocyte surface.
Location Bias Contributes to Functionally Selective Responses of Biased CXCR3 Agonists to Regulate Inflammation
Authors: Gardner Julia , Eiger Dylan , Boldizsar Noelia , Honeycutt Christopher C. , Kirchner Stephen , Hicks Chloe , Choi Issac , Zheng Kevin , Warman Anmol , Smith Jeffrey , Zhang Jennifer , Rajagopal Sudarshan
Published: May 2022

G Protein‐Coupled Receptors (GPCRs) represent the largest and most diverse family of cell receptors in the human genome and are targeted by ~35% of all FDA‐approved drugs. GPCR signaling is mediated by various effectors, including G proteins, β‐arrestins, and GPCR kinases (GRKs). Recent studies have uncovered that different ligands can bind to the same GPCR and differentially activate specific signaling pathways, a phenomenon known as biased agonism. Most research has focused on characterizing biased agonism at the plasma membrane, but GPCRs are also known to traffic to and signal from a variety of subcellular compartments. To determine if GPCR subcellular localization contributes to biased signaling, we studied how the chemokine family GPCR CXCR3 signals from endosomes following stimulation by the three endogenous ligands CXCL9, CXCL10, and CXCL11.

We determined that the biased ligands of CXCR3 promote different amounts of receptor mediated endocytosis and also show markedly different G protein and β‐arrestin signaling profiles at both the plasma membrane and the endosome. The biased activation of G proteins and β‐arrestins promotes differential activation of cytoplasmic and nuclear ERK1/2, as well as cellular transcription in both HEK293 cells and primary CD8+ T‐cells. Inhibition of receptor internalization greatly diminishes the biased signaling observed between the endogenous chemokines. Finally, we demonstrate in a murine model of contact hypersensitivity that receptor internalization is a key component of the ability of a synthetic β‐arrestin biased CXCR3 agonist to potentiate the inflammatory response. The present study shows the CXCR3 biased ligands induce unique signaling profiles at the plasma membrane and the endosome, and that signaling from endosomes greatly contributes to the overall biased responses seen at CXCR3. These results demonstrate that signaling from subcellar compartments is a critical component of GPCR signaling. Our findings have implications in the development of pharmaceutical drugs targeting chemokine receptors and other GPCRs.
The Role of Monounsaturated Fatty Acids in Ferroptosis
Authors: Pope Lauren E. , Dixon Scott
Published: May 2022

Exogenous monounsaturated fatty acids (MUFAs) such as oleate (18:1 n‐9) protect cells from ferroptotic cell death, contributing to cancer cells’ metastatic potential in vivo. Understanding how MUFAs prevent ferroptosis is critical for developing new anti‐cancer therapeutics that target this non‐apoptotic cell death process. A diversity of MUFAs with varying chain lengths and double bond positions are synthesized in human cells and found in human diets. It is presently unclear which MUFAs can inhibit ferroptosis, and whether all those that are protective act through the same mechanism. To investigate, we have screened a library of structurally distinct MUFA species for their ability to inhibit ferroptosis under various conditions. We find that protection against ferroptosis is MUFA structure‐specific, with only certain species being able to protect from ferroptosis. Further investigation indicates that, in response to a pro‐ferroptotic stimulus, protective MUFAs can inhibit the formation of toxic lipid reactive oxygen species while non‐protective MUFAs are unable to do so. Interestingly, some non‐protective MUFAs nonetheless appear to enter the cells and become activated. Thus, we posit that enzymes necessary for MUFA‐mediated ferroptosis protection specifically activate and incorporate only certain protective MUFAs into membrane phospholipids. Further characterization of the MUFA metabolic pathway will inform endeavors to synthesize potent ferroptosis inducers that overcome the protective nature of MUFAs in ferroptosis in vivo.
Metabolic and Cardiac Manifestations in a Mouse Model of Genetic Mitochondrial Hepatopathy Without Obesity: Evidence for a Sexual Dimorphism
Authors: Burelle Charlotte , Deschênes Sonia , Cuillerier Alexanne , De Loof Marine , Higgins Marie‐Eve , Clavet Marie‐Elaine , Daneault Caroline , Sirois Martin , Burelle Yan , Ruiz Matthieu
Published: May 2022

Objective

In non‐alcoholic fatty liver disease (NAFLD), liver mitochondrial dysfunction plays an important role in the development of cardiac abnormalities independent of obesity per se. However, the mechanisms underlying the development of these abnormalities, and whether these are affected by sex remain unclear. In the present study, we sought to address this question using a mouse model of hepatic mitochondrial deficiency caused by loss of Lrpprc (H‐Lrpprc‐/‐) as these mice develop microvesicular steatosis and a NAFLD lipidomic‐like profile without obesity.

Hypothesis

Our working hypothesis is that metabolic defects in the liver would trigger metabolic and functional abnormalities in the heart in a sex‐dependent manner.

Methods

Detailed phenotyping experiments were performed in male and female H‐Lrpprc‐/‐mice and their controls at 14 weeks of age. Glucose metabolism was monitored by blood glucose as well as insulin, and glucose tolerance tests (ITT, OGTT). Cardiac manifestations were studied using molecular (qPCR), lipidomic (mass spectrometry) and functional (oximetry on isolated mitochondria, intraventricular pressures by Millar probe) analyses.

Results

Genetic inactivation of Lrpprc resulted in an >60% reduction of LRPPRC protein content in both males and females, which resulted in comparable structural liver abnormalities. In contrast, the resulting metabolic and cardiac functional changes displayed a marked sexual dimorphism. Lrpprc deficiency in the liver was associated with fasting hypoglycemia and increased insulin sensitivity in both sexes, but more so in males. Lrpprc deficiency was also associated with a remodeling of specific markers of mitochondrial function exclusively in the female myocardium. This included 1) reduced expression of gene markers for mitochondrial biogenesis (Tfam), fusion (Mfn1‐2), and fatty acid (FA) utilization and oxidation (Pparα, Cd36, Cpt1b, Cpt2, Vlcad, Lcad, Mcad), 2) impairment of FA‐dependent mitochondrial respiration, and 3) increased cardiac triglyceride levels. Paradoxically, cardiac function was preserved in females, while males displayed mild diastolic dysfunction as evidenced by increased early and late diastolic pressures (0.29 and 2.39 fold vs. male wild type respectively) amongst other parameters.

Conclusion

Collectively, these results indicate that metabolic defects in the liver can result in significant sex‐dependent abnormalities that affect both the mitochondrial/metabolic phenotype and contractile function independent of obesity. This experimental model may prove useful to better understand the mechanisms underlying the sex‐related variability in the progression of lean NAFLD in humans.
Novel roles of tendon in the postnatal growth and expansion of TMJ condyle
Authors: Jing Yan , Ma Chi , Liang Alice , Feng Jian Q.
Published: May 2022

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well‐established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X‐ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum‐formed bone (EFB, Fig.1c1), cartilage‐formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum‐formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle‐shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin‐4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx‐CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx‐CreERT2; R26RDTA (carrying a loxP‐flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).
Dissecting alveolar patterning and maintenance at single‐cell resolution
Authors: Gillich Astrid , Brownfield Douglas G. , Travaglini Kyle J. , Zhang Fan , Farmer Colleen G. , St. Julien Krystal R. , Tan Serena Y. , Gu Mingxia , Zhou Bin , Feinstein Jeffrey A. , Metzger Ross J. , Krasnow Mark A.
Published: May 2022

In mammalian lungs gas exchange occurs in thin‐walled air sacs called alveoli, which are surrounded by a dense mesh of capillaries. Defects in patterning, maintenance or repair of alveoli lead to diseases that compromise gas exchange, including chronic diseases such as bronchopulmonary dysplasia, pulmonary fibrosis and chronic obstructive pulmonary disease, as well as the acute respiratory distress syndromes accompanying severe alveolar injury or virus‐induced damage, as in Covid‐19. Despite the tremendous disease burden and the urgent need for therapies, the mechanisms that establish and maintain the pattern and architecture of alveoli are not well understood. Here we use mosaic genetic labeling, single‐cell RNA‐sequencing and high‐resolution deep imaging to elucidate the three‐dimensional structure and cellular composition of alveoli. We show that an alveolus in the mouse lung is composed of 10‐15 cells of seven different types, each with a remarkable, distinctive structure. Two of them are intermingled capillary cell types with complex ‘swiss cheese’ morphologies and distinct functions. One cell type that we name the ‘aerocyte’ is specialized for gas exchange and unique to the lung. The other cell type, termed ‘general capillary’, is specialized to regulate vasomotor tone and functions as a progenitor cell in capillary maintenance and repair. By mapping alveolar development at single‐cell resolution at a defined position in the lung, we find that alveoli form surprisingly early by budding of epithelial cells out from the airway stalk between enwrapping smooth muscle cells that rearrange into a ring of myofibroblasts at the alveolar entrance. Our analysis suggests a novel mechanism of alveolar formation and provides the foundation for investigations of the structure, function and maintenance of the gas exchange surface in health, disease, aging and evolution.
Characterization of RTK Tyrosine Phosphorylation in Response to Chemotherapy Drugs in Triple Negative Breast Cancer
Authors: Flores Perez Andrea S. , Conage‐Pough Jason E. , White Forest
Published: May 2022

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is characterized by the absence of receptors commonly found in breast cancer. Due to the lack of critical biomarkers, there are no targeted therapies for TNBC and as a result TNBC cases tend to have poorer prognosis and shorter relapse‐free survival than other types of breast cancer. One of the most well‐established therapeutic targets in breast cancers are receptor tyrosine kinases (RTKs). In TNBC, about half of the cases overexpress a subtype of RTKs called the epidermal growth factor receptor (EGFR) family. However, the outcome of EGFR‐targeted therapies in TNBC patients have been disappointing due to lack of established biomarkers that indicate sensitivity to EGFR inhibitors in patients. Previous studies have shown that studying tyrosine phosphorylation (pTyr) in signaling pathways can be quantitated to measure unique dysregulation of RTK signaling networks in patient samples. In this study, we use liquid chromatography‐mass spectrometry (LC‐MS) techniques to identify and quantify pTyr in TNBC in response to the following chemotherapy treatments: Taxol (genotoxic) and Erlotinib (EGFR inhibitor). We evaluated viability of TNBC cells in‐vitro­in response to Taxol and Erlotinib, where Erlotinib appeared to have an antagonist effect when paired with Taxol. LC‐MS results provided further evidence of the antagonist effect of Taxol and Erlotinib in the following markers: EGFR, AURKB, ITGA3, and ITGB1. These results could reveal potential biomarkers that indicate tumor sensitivity to targeted therapies in TNBC.
Infusion of C20 ceramide induces depressive‐like behavior, increases microglia but not astrocyte expression
Authors: Fonseca‐Ferrer Wilfred , Suárez‐Gomez Cristina , Sambolin‐Escobales Lubriel , Santos‐Avilés Kimberly , Colón‐Romero Maria , Hernández‐López Anixa , Feliciano‐Quiñones Adariana , Tirado‐Castro Lizmarie , Porter James
Published: May 2022

Depression is a disease affecting the population worldwide. Depressed patients have shown alteration in the hippocampus suggesting this region can be an important region to study the impact of the development of depressive‐like behavior. Literature has demonstrated depressed patients present an increased levels of long‐chain ceramides. Ceramide is a sphingolipid component of the cellular membrane and play an important role in its structural integrity. To better understand depression mechanisms it is important to assess the glial cells mechanisms, since these cells play an essential role in inflammation, which is increased in depressed patients. Literature has reported interaction between astrocytes and microglia, still their detailed mechanism, their individual response to ceramide signaling, and contribution to depressive‐like behavior development are not clearly understood. Some research has demonstrated that microglia are activated in the presence of C20 and C16 ceramide. Otherwise, data have shown that reactive astrocytes interact with C16 ceramide but not with C24 ceramides. Because of this, we hypothesize that the infusion of long‐chain ceramides (C20) in the ventral hippocampus will increase the development of depressive‐like behavior, will cause an increase in the level of microglia expression but not astrocyte expression. We performed seven infusions of long‐chain ceramides into the ventral hippocampus followed by behavior analysis to measure the development of depressive‐like behavior and tissue collection to analyze inflammatory profile. Results show that rats infused with long‐chain ceramides present development of short‐term anhedonia‐like behavior twenty‐four hours after infusions but no depressive‐like behavior was assessed five days after the last infusion of ceramides. Also, infusion of ceramides induce an increase in microglia expression but no significant differences were assessed in astrocyte expression. These results suggest that C20 ceramide activates the microglia inflammatory pathway but not astrocyte mediated inflammation. There might be different mechanisms inducing inflammation in microglia and astrocytes. This preliminary data can also suggest that microglia mediated inflammation can contribute more to depressive‐like behavior than astrocytes. These results bring speculative significance, but more comprehensive studies can show a higher understanding of ceramide signaling in inflammation and a better understanding of the contribution of microglia and astrocytes to the development of depressive‐like behavior.
The impact of hydro‐dissection method in fascial plane delineation of dorsum of human hand
Authors: Kumar Madan
Published: May 2022

In recent Ayurveda Anatomy education, hydro‐dissection method (HDM) has made technical advancements. It has been introduced as part of regular Ayurveda Anatomy lab. The author undertook investigation of dorsum of an embalmed human hand, using HDM, to visualise and analyse the intricacies of fascial components.

Designing and assembling of the hydro‐dissection unit: An instrumentation is prepared, simulating the Sushruta Samhita mentioned method of dissection, for the concerned study. The instrument maintains an optimal standard for hydro‐dissection, monitoring and maintaining the temperature, slipstream, pH of the water and asepsis. The instrument also facilitates adequate visualization of the specimen without compromising the austerity of the procedure.

Hydro‐dissection method and hydro‐dissection instruments: A blunt dissection is done using brushes and scrapers of various dimensions which help in segregation and differentiation of structures seen during HDM. Photos and videos along with textual documentation will be performed during the entire duration of each session. Identification and confirmation of the structures will be done by experts. Side‐by‐side comparison with existing atlases will be also done.

The dorsal fascia of the hand is situated on the dorsal tendons of dorsal aspect of hand. The superficial fascia or the subcutaneous tissue is described as a layer of loose areolar tissue or adipose tissue. The difference in the thickness of fascia of palmar and dorsal hand is attributed to the difference in the range of mobility of structures passing through these areas. In this experiment, the author has tried to determine the relations in the transitional zones between the mid hand and the fingers by a layer‐by‐layer dissection. The superficial fascia is identified as a double layer of superficial fat and deeper fibrous tissue. It is separated from deeper layers by loose areolar tissue. The deep fascia becomes extremely thin distally. Usually, when the deep fascia becomes thin, it cannot be prepared as a continuous layer beyond the mid hand. As it passes distally, the fascia tends to blend with the superficial fascia. In the current scenario, the author has identified remarkable progress in the dissection, using HDM by delineating various planes of the fascia along with the resident and traversing structures.
Unique structural and dynamic properties of the HNH nuclease in mesophilic and thermophilic Cas9 revealed by NMR
Authors: Belato Helen , D’Ordine Alexandra , Jogl Gerwald , Lisi George
Published: May 2022

CRISPR‐Cas9 is a widely utilized biochemical tool with applications in biotechnology and in medicine. Target DNA recognition and nuclease sites of Cas9 are spatially separated yet functionally coupled, indicative of an allosteric crosstalk throughout the enzyme. There are structural and dynamic changes occuring throughout Cas9 that are critical for this signal propagation. However, an understanding of the molecular motions guiding Cas9 and how this influences its mechanism are unknown. In this study we report on the structural and dynamic properties of the HNH domain between the extensively studied SpCas9 from Streptococcus pyogenes, and a recently discovered thermostable Cas9 homolog from a thermophilic bacterium Geobacillus stearothermophilus(GeoCas9). Here we show that despite sequential and structural conservation, SpHNH and GeoHNH have different dynamic profiles. We found the intrinsic dynamics of SpHNH to be on the slow (ms) timescale, while GeoHNH is highly influenced by fast (ps‐ns) timescale dynamics. Furthermore, we found that when the residue with the highest flexibility on the ps‐ns timescale is mutated in GeoHNH, its protein solubility, thermal stability, and dynamic profile are all drastically distorted. Our results demonstrate how there are mechanistic differences between the HNH domain of mesophilic and thermophilic Cas9 species. Our results also highlight how these fast timescale motions that regulate GeoHNH play a role in giving GeoCas9 its thermophilic characteristic.