bluuebunny/crossref_metadata_2025 huggingface dataset:
Misfolding Propensity of SAA1 Fragments: Special Focus on the Signal Peptide Region
Authors: Haines Morgan S. , Pirela James P. , Nylen Kendra N. , Fortin Jessica S.
Published: May 2022

AA amyloid is the most common form of systemic amyloidosis reported in human medicine and across animal species. AA amyloidosis is the result of over‐production by hepatocytes and aberrant processing of acute‐phase serum amyloid A1 (SAA1). Proteolytic cleavage of SAA1 is believed to play a central role in AA amyloid formation. The SAA1 protein is of 122 amino acids (aa) and undergoes a first cleavage of 18 residues consisting of the signal peptide at the N‐terminal region. The aggregation propensity of the signal peptide region has never been studied. To better understand the mechanism behind systemic amyloidosis in the SAA1 protein, we studied the misfolding propensity of the signal peptide region considering that SAA1 is going through further cleavage processes. The goal of this research was to determine the propensity of the SAA1 signal peptide region to generate fibrils. We examined the signal peptide amino acid sequence of serum amyloid A1 (SAA1) derived from different animal species. A library of the 25 peptides was designed to study the fibril formation using in silico analysis and in vitro assays. The amyloidogenic potential of each SAA1 signal peptide homolog was assessed using thioflavin T fluorescence assays, Congo red binding assays, and transmission electron microscopy. After 7 days of incubation, 22 SAA1 signal peptide fragments had the propensity to form fibrils at a concentration of 500 µM with 50% hexafluoroisopropanol at 37 ºC. The signal peptide region might be important in contributing to the misfolding of aggregation‐prone proteins. Characterizing these conformations are relevant as they may be involved in seeding intact and/or fragmented SAA1, contributing to mechanism of misfolding. This information could be important for continuation studies in the understanding of misfolding disorders.
Palmaris Longus Inversus Muscle Present Bilaterally in an 82‐Year‐old Cadaver and Unilaterally in a 68‐Year‐old Cadaver
Authors: Granite Guinevere , Wind Gary , Leighton Maria X.
Published: May 2022

The palmaris longus muscle is one of the most variable muscles in the human body. Palmaris longus muscle anatomical variations include reversed (palmaris longus inversus muscle), duplicated, triplicated, bifid, hypertrophied, accessory (additional) slips, and/or complete absence. During anatomical dissection of fifty cadavers in the 2019 undergraduate first‐year anatomy course at the Uniformed Services University of the Health Sciences, we found a palmaris longus inversus muscle bilaterally on an 82‐year‐old white female cadaver and a unilateral palmaris longus inversus muscle on the right forearm of a 68‐year‐old white female cadaver. Due to the prevalence of anatomical variations in the palmaris longus muscle, adequate knowledge of such prevalence and clinical significance is necessary in various medical specialties for its use in reconstructive surgery, and the possibility and treatment of clinical syndromes. An accessory (additional) palmaris longus muscle, hypertrophied palmaris longus muscle, hypertrophied palmaris longus inversus muscle, or just the presence of a palmaris longus inversus muscle, can all cause compression to adjacent neurovascular structures, such as the median nerve, ulnar nerve, ulnar artery, and/or the anterior interosseous artery, in the distal forearm and wrist. Palmaris longus tendons are used as grafts in a variety of surgeries, such as extensor tendon repair in rheumatoid arthritis patients, injuries of flexor tendons and repair, lip augmentation, reconstructive hand surgery, frontalis suspension sling in ptosis correction, plastic surgery, and pulley reconstructions. Surgeons, physicians, radiologists, and physiotherapists must be knowledgeable of the many palmaris longus muscle anatomical variations and their possible contributions to pathological processes.
Hypertension Induces Contractile Dysfunction in Rat Mesenteric Lymph Vessels
Authors: Pal Soumiya , Henry David , Rhee Sung W. , Stolarz Amanda J.
Published: May 2022

Hypertension predisposes patients to a cluster of deadly cardiovascular and metabolic diseases involving inflammation, dyslipidemia, and fluid imbalance. Current medications fail to normalize blood pressure in 25% of hypertensive patients; therefore, we considered the possibility that the lymphatic circulation provides a new therapeutic target to alleviate hypertension and its associated co‐morbidities. The intrinsic driving force for the propulsion of lymph fluid is the robust, spontaneous rhythmic contractions of lymphatic muscle cells (LMCs) that facilitate the lymph flow which helps maintain fluid balance and the transport of lipids and immune cells. Thus, evaluation of lymphatic contractions and estimation of lymph flow during hypertension is important to understand the possibility of targeting lymphatic circulation in hypertension. Angiotensin II (Ang II) is a key regulator of blood pressure, and increased levels of Ang II are known to induce hypertension. However, the role of Ang II mediated hypertension on lymphatic contractile dysfunction is still a matter of debate. Here, we evaluated the rhythmic contractions of LVs isolated from Ang II infused hypertensive rats (AHT LVs) compared to normotensive controls (NT LVs). Ang II containing osmotic mini pumps (500 ng/kg/min) were implanted subcutaneously in adult (9‐13 weeks old) male Sprague Dawley (SD) rats for 14 days. Rat mesenteric LVs were dissected free from fat and then cannulated and pressurized (4‐5 mmHg) in a perfusion bath filled with physiological salt solution (PSS). Changes in diameter were monitored by real‐time edge detection software. Baseline rhythmic contractions were recorded for ~45 minutes and then a L‐type calcium channel antagonist, nifedipine (NIF; 1 µmol/L) was added to the bath for 10 mins followed by washout with calcium free PSS. AHT LVs had significantly increased calcium dependent tone to 14.3% ± 5.1% (n=8) compared to NT LVs, which remained at 5.2% ± 1.1% (n=8). AHT LVs also exhibited a larger response to nifedipine with 14.0% ± 6.5% (n=6) increase in dilation compared to NT LVs 3.9% ± 0.9% (n=8). Contractile traces also showed visible dysrhythmia, defined as an increased variation in contraction interval, in LVs isolated from AHT rats at 32.4% ± 14.5% (n=8) when compared with NT LVs at 2.3% ± 1.1% (n=8). An increased variance for the time to relaxation also was observed in AHT LVs (7.4% ± 4.6%; n=8) compared to NT LVs (0.6% ± 0.2%; n=8). These differences ultimately resulted in a significant decrease in calculated flow in AHT LVs (62.0 nL/s ± 8.4 nL/s; n=5) compared to NT LVs (92.4 nL/s ± 4.3 nL/s; n=5). Our data suggest that Ang II mediated hypertension can cause lymphatic contractile dysfunction leading to loss of lymph flow and may be a potential mechanism by which hypertension increases the risk of associated co‐morbidities. Studies are ongoing to assess the changes in lymph flow in vivo during Ang II mediated hypertensive versus normotensive rats.
Developing A Reference Charts for Foetal Biometry in Indian Population to Predict Growth & Development of Foetus
Authors: Agrawal Dushyant , Mishra Sanjay , Ghatak Surajit , Singh Pratibha , Garg Pawan K.
Published: May 2022

Objectives

The objective of the study was to develop reference charts for foetal abdominal circumference and femur length in normal singleton pregnancies. The modality used in the study was by means of ultrasonography which is easily available and safe during the pregnancy.

Method

In this study, we conducted ultrasonography examination in 300 singleton pregnant women coming for routine checkups. Out of various foetal biometric parameters abdominal circumference (AC) and femur length (FL) were measured and reference charts (10th, 50th, 90th and 95th percentiles) for each parameter corresponding to its gestational age were developed.

Results

Age distribution of pregnant women was uniform (p=0.87). Both parameters i.e. AC and FL were found to be in linear correlation with the corresponding gestational age in weeks. Therefore, their use in determining development and age of the foetus is reliable. We compared our reference charts with other studies conducted in European and Chinese population. From which, it is obvious that the data found in Indian population is different from studies conducted in other ethnic population.

Conclusion

Reference charts derived in this study can be used to determine growth and development of foetus depending upon Indian standards. Because ethnicity, race, nutritional and environmental factors are known to impact the foetal development. Most of the health care set ups are using data from the studies conducted in western population. Which may cause misrepresentation of data in diagnosis. Thus can lead to unnecessary medical intervention which may cause burden in limited resources in developing nations like India. Therefore, we conclude that each population should have their own reference charts for foetal biometry.
Diabetes Mellitus Drug Discovery: Insights into Targeting Feline and Human Amylin with Small Molecules
Authors: Smith Alyssa A. , Duggan Kelsey E. , Fortin Jessica S.
Published: May 2022

In a majority of diabetic feline and human patients, amyloid deposits have been detected in the islets of Langerhans. These deposits originate from islet amyloid polypeptide (IAPP or amylin), a satiety hormone produced and co‐secreted with insulin by beta‐cells. These islet amyloid deposits have been associated with beta‐cell death during the progression of diabetes. Several molecular entities have been shown to inhibit human IAPP aggregation, including silibinin and resveratrol. However, these agents have poor bioavailability and cause multiple pharmacological effects. An effective means to stop or prevent pancreatic amyloidosis in diabetes mellitus with small drug‐like molecules is still not available. The overall objective of this project was to identify selective and general inhibitors of feline and human IAPP fibril formation. The potency of molecules to reduce the formation of feline and human IAPP fibrils and toxic oligomers was monitored in vitrousing biophysical methods such as Thioflavin T (ThT) fluorescence assays, dynamic light scattering (DLS), and transmission electron microscopy (TEM). We then assessed the potency of the JF analogs on both feline and human IAPP, which differ by only 4 amino acids. JF‐19‐033 selectively inhibited feline IAPP fibril formation. JF‐19‐034 selectively inhibited human IAPP fibril formation. In contrast, JF‐19‐029 non‐selectively inhibited all of the human and feline IAPP peptides we tested. By doing this, we identified selective and non‐selective inhibitors of IAPP fibril formation. This project represents a phenotypic‐based drug design strategy for modulating feline and human IAPP fibril formation based on amino acid variations by small molecules. As a result, it has the potential to point towards new therapeutic strategies for type 2 diabetes.
A Phenotypic‐based Drug Discovery Approach to Inhibit the Misfolding of Hyperphosphorylated Tau Isoform 1N4R
Authors: Fortin Jessica S. , Wang Kuang‐Wei , Chien Hsiao T. , Kuo Min‐Hao
Published: May 2022

The vast majority of Alzheimer’s disease (AD) drug development efforts have failed because of significant gap in the understanding of the pathophysiological processes and insufficient effort in identifying, optimizing, and validating small molecules inhibitors of aggregation. AD involves the misfolding of amyloid‐beta which forms Aβ plaques and, subsequently, activates different types of microglial cells leading to a pro‐inflammatory state. Microglia when activated have been known to increase tau phosphorylation and drive the trans‐synaptic spread of aggregated hyperphosphorylated tau (p‐tau). Most importantly, the spatiotemporal distribution of neurofibrillary tangles predicts cognitive impairment. Instead of the unmodified tau that shows minimal effects on cell viability, our research focuses on the control of p‐tau that possesses disease‐relevant molecular features, including hyperphosphorylation and cytotoxicity. Our drug discovery project is centered on the isoform 1N4R due to its prevalence (1N) and long‐distance trans‐synaptic propagation (4R). We identified compounds that inhibit the aggregation and cytotoxicity of p‐tau, which represent a novel approach for AD drug discovery and development. Due to lack of binding pocket(s) and fast‐switching conformation of p‐tau, we opted for a phenotypic‐based drug screening approach to identify and optimize hit candidates. In our current project, the anti‐aggregation activity and cellular protection of newly synthesized small molecules were evaluated with biophysical methods (thioflavin S fluorescence assays, photo‐induced cross‐linking of unmodified proteins, dynamic light scattering) and survival assays with human neuroblastoma SH‐SY5Y cells. We prepared and tested 3 different series of small molecules and challenged these molecules with our first‐tier screening assay, i.e. thioflavin S fluorescence assays. Ureas and sulfonylureas series failed to inhibit the aggregation of p‐tau 1N4R. The best prototypical representative of the sulfonamide series, compound JF‐19‐73, effectively antagonized the cytotoxicity of p‐tau in vitro at low micromolar concentrations, suggesting the potential for neuroprotection in vivo. Other representatives of the sulfonamide series exhibited an intermediate effect to antagonize the aggregation and cytotoxicity of p‐tau isoform 1N4R. This project opens doors in targeting undruggable prone‐to‐aggregate proteins such as p‐tau and provides a molecular scaffold for further optimization and pre‐clinical studies focused on AD drug development.
A Role of NOD2 in CARTp‐mediated Mechano‐hypersensitivity
Authors: Schafer Rachel , Giancotti Luigi , Chen Zhou , Doyle Tim , Harada Caron , Zhang Jinsong , Salvemini Daniela
Published: May 2022

Neuropathic pain is a debilitating chronic condition that remains difficult to treat. There is a high priority to identify novel non‐opioid‐based therapeutic targets as long‐term use of opioids is problematic due to its severe side effects and strong abuse potential. Our lab recently discovered G‐protein coupled receptor 160 (GPR160) has a role in neuropathic pain. Gpr160 was upregulated in the dorsal horn of the spinal cord (DH‐SC) on the side of nerve injury relative to the uninjured side in mice. Blocking GPR160 using siRNA or a neutralizing antibody (Ab) reversed and prevented pain hypersensitivity. In addition, our lab deorphanized GPR160 as the receptor for cocaine‐ and amphetamine‐regulated transcript peptide (CARTp). We showed that an intrathecal (i.th.) injection of CARTp in naive mice caused mechano‐hypersensitivity that was dependent on GPR160. CARTp‐induced gene expression is relatively unexplored and the mechanism by which CARTp/GPR160 signaling promotes chronic pain is not well known. Therefore, our objective was to perform an unbiased RNA transcriptomics analysis to identify which genes were altered at the time of CARTp‐mediated peak pain in the DH‐SC. We found that Nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2) expression was increased upon i.th. injection of CARTp and its expression decreased after CARTp was co‐administered with GPR160 Ab, pointing to a potential interaction between CARTp/GPR160 and NOD2. NOD2 is a cytosolic pattern recognition receptor that is involved in activating the immune system in response to pathogens. Moreover, a recent study linked NOD2 to the development of neuropathic pain. This led to our hypothesis that CARTp/GPR160 causes mechano‐hypersensitivity through NOD2 and NOD2 has a functional role in neuropathic pain. C57BL/6 (WT) and NOD2‐/‐ mice received an i.th. injection of CARTp and mechano‐allodynia was assessed. The WT mice developed mechano‐allodynia by 30min and persisted for 5hrs. In contrast, mechano‐allodynia was attenuated for 4hrs in NOD2‐/‐ mice, suggesting CARTp/GPR160 induces mechano‐hypersensitivity through NOD2. Furthermore, we found that NOD2 has a functional role in a chronic constriction injury (CCI) model of neuropathic pain. WT mice started developing mechano‐allodynia on day 3 (D3) after CCI, reached a maximum by D7, and persisted until D14. However, NOD2‐/‐ mice did not develop mechano‐allodynia until D10, indicating that NOD2 is involved in the development of CCI neuropathic pain. Overall, our results provide a potential mechanistic insight on how CARTp causes mechano‐sensitivity and NOD2 has a functional role in CCI‐mediated neuropathic pain.
Elevated 5‐hydroxytryptamine in COVID‐19 Stimulates ANO1 Mediated Cl Secretion in Lung & Intestinal Epithelial Cells
Authors: Hoque Kazi M. , Hayashi Mikio , Sheikh Irshad A. , Banerjee Aditi , Verma Subhash C. , Leblanc Normand , Zeiss Caroline J. , Ameen Nadia , Chakraborty Subhra
Published: May 2022

Earlier studies demonstrated that blood 5‐hydroxytryptamine (5‐HT) is elevated in patients with COVID‐19‐associated diarrhea with higher severity of symptoms. We hypothesize that disruption of vectorial Cl transport by 5‐HT may be critical in determining the alveolar flooding and abnormalities in intestinal Cl secretion through stimulation of anoctamin 1(ANO1) Cl channel. Using western blot, immunostaining, and electrophysiology, we characterized the localization of human ANO1 and its stimulation by 5‐HT on Cl secretion in lung and intestinal epithelium. Because calcium‐activated chloride current in human intestinal epithelia remains controversial, we examined the localization of ANO1 in the human terminal ileum and colonic tissue using confocal microscopy. Our results indicated that ANO1 was localized predominantly at the brush‐border membrane and co‐localized with the brush‐border membrane marker villin. ANO1 is not present in goblet cells. The anti‐ANO1 antibody recognized a protein of appropriate size in human colonic tissue. The specificity of the ANO1 antibody was tested by immunoblot analysis of lysates from human colorectal cancer tissues, where it displayed amplified ANO1 protein expression. We next confirmed ANO1‐currents activated by 5‐HT in the Caco‐2 cells by patch‐clamp measurements of whole‐cell current. The application of 100 nM 5‐HT produced a typical outward rectification. CaCCinh‐A01, a specific ANO1 blocker, inhibited the currents. The half‐maximal effective concentration value for the effects of 5‐HT was estimated at 21.8 ± 13.7 nM with a Hill coefficient of 0.89 ± 0.16. These results indicated that 5‐HT evoked calcium‐activated Cl currents through ANO1 channels. ANO1 is expressed in Calu 3 cells. We next confirmed the presence of ANO1 currents activated by 5‐HT in Calu‐3 cells by the Ussing chamber experiments. Serosal addition of 5‐HT produced an immediate and significant increase in Isc in Calu‐3 cells that was inhibited by the ANO1 selective inhibitor T16Ainh‐A01. Finally, we demonstrate that SARS‐CoV2 infection led to enterochromaffin cell hyperplasia in the intestinal epithelium of Syrian Hamster with a possible elevation of 5‐HT, which could explain the severity of symptoms in COVID‐19 associated diarrheal patients.

In conclusion, SARS‐CoV2 infection resulted in intestinal enterochromaffin cells hyperplasia that could elevate 5‐HT. Elevated 5‐HT activates luminal ANO1 CaCC in the intestinal and lung epithelium by a mechanism that appears to involve the rise of [Ca2+]i. Our data suggest that 5‐HT may be a critical determinant of the COVID‐19 associated diarrhea and flooding of alveoli that have considerable implications for COVID‐19 therapy.
Critical Environmental Limits for Young, Healthy Adults (PSU HEAT)
Authors: Wolf S. T. , Cottle Rachel M. , Vecellio Daniel J. , Kenney W. L.
Published: May 2022

The average temperature of the Earth has been steadily increasing for decades and is expected to continue increasing throughout the 21st century. Along with the increase in average global temperatures, the frequency, duration, and severity of heat waves have increased. Thus, there is a critical need for research targeted at understanding the specific environmental conditions associated with increased risk of heat‐ and humidity‐related morbidity and mortality.

Purpose

To establish critical environmental limits for healthy young adults at two metabolic rates that reflect activities of daily living and light activity. We hypothesized that critical environmental limits would be shifted toward lower combinations of temperature and humidity at higher compared to lower rates of metabolic heat production.

Methods

Twenty‐five (12M/13F; 24 ± 4 yrs) subjects were exposed to progressive heat stress in an environmental chamber at two rates of metabolic heat production chosen to represent minimal activity (MinAct) or light ambulation (LightAmb). Progressive heat stress comprised either (1) constant dry‐bulb temperature (Tdb; 36, 38, or 40 °C) and increasing ambient water vapor pressure (Pa; Pcrit trials), or (2) constant Pa (12, 16, or 20 mmHg) and increasing Tdb (Tcrit trials). Chamber Tdb and Pa, and subject gastrointestinal temperature (Tgi), were continuously monitored throughout each trial. After a 30‐min equilibration period, progressive heat stress superseded until subject heat balance could no longer be maintained and a clear rise in Tgi was observed. Each subject was tested during MinAct and LightAmb in 2 or 3 randomized environmental conditions, for a total of 4–6 trials per participant.

Results

By design, oxygen consumption (V̇O2: 0.46 ± 0.10 vs. 0.80 ± 0.16 L/min; 1.8 ± 0.3 vs. 3.2 ± 0.3 METs), metabolic heat production (82.9 ± 12.5 vs. 133.3 ± 14.8), and sweat rate (142.8 ± 80.2 vs. 230.8 ± 84.9) were lower for MinAct compared to LightAmb (all p < 0.0001). There were no differences in these variables (p ≥ 0.05) among environmental conditions for either MinAct or LightAmb intensities. Higher metabolic heat production (p < 0.001) during LightAmb compared to MinAct trials resulted in significantly lower critical environmental limits across all Pcrit and Tcrit conditions (all p < 0.001).

Conclusions

These data are the first to define critical environmental limits for the maintenance of heat balance in healthy young adults during minimal to light physical activity reflecting activities of daily living and light household tasks. By establishing critical environmental limits in a young, healthy population, the findings of this study will be used as a benchmark for future studies in aged and other vulnerable populations, and can be used to develop guidelines, policy decisions, and evidence‐based alert communications to minimize adverse impacts of extreme heat events.
Differential Effects of Vascular Endothelial Growth Factor on Glycocalyx of Endothelial and Tumor Cells Uncovered by Super‐resolution Optical Microscopy and Potential Targets for Tumor Metastasis
Authors: Fu Bingmei , Xia Yifan , Li Yunfei
Published: May 2022

On the surface of every mammalian cell, there is a matrix‐like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by vascular endothelial growth factor (VEGF, VEGF‐A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. But how the TC secretion VEGF affects its own GCX is unknown although bulkier GCX of tumor cells was found to enhance its metastasis. To investigate the VEGF effect on TC GCX and to elucidate the ultrastructural organization of EC and TC GCX and their alteration by VEGF, we employed super‐resolution stochastic optical reconstruction microscopy (STORM) to observe the spatio‐chemical organizations of the heparan sulfate (HS) and hyaluronic acid (HA), two representative GAGs of GCX, on human cerebral microvascular endothelial cells (hCMEC) and malignant breast cancer cells MDA‐MB‐231 (MB231). We found that HS and HA have distinct organizations on hCMEC and MB231. Only HS of hCMEC is perpendicular to the cell surface, while HA of hCMEC, as well as HS and HA of MB231 all lie in the same plane as the cell surface where they appear to weave into a 2D network covering the cell. We also found that VEGF significantly reduces the length and coverage of HS on hCMEC but does not change the thickness and coverage of HA on hCMEC. On the contrary, VEGF significantly enhances the coverage of HS and HA on MB231 although it does not alter the thickness. The differential effects of VEGF on the GCX of TC and that of EC may favor TC adhesion and transmigration across EC barriers for their metastasis.
Insulin Signaling Suppresses Autophagy in Astrocytes
Authors: Geffken Shawn , Smith Catherine , Lee Hiu H. , Cai Weikang
Published: May 2022

Insulin is a peptide hormone produced in the pancreas that is crucial in regulating systemic glucose homeostasis and energy balance. Diabetes mellitus is an endocrine disease that can be broken down into two subtypes. Type I diabetes is low to non‐measurable amounts of insulin being produced by the pancreas consequent to cellular damage. Type II diabetes is caused by insulin resistance and later, decreased insulin secretion. Both conditions lead to hyperglycemia and impaired insulin signaling throughout the body if left untreated. Numerous studies have shown that insulin signaling controls several metabolic pathways in peripheral insulin‐sensitive tissues, such as liver, adipose tissue, and muscle. Among them, autophagy is one of the most potent pathways suppressed by insulin. Autophagy is a molecular mechanism by which cells recycle the damaged or outdated components, including proteins and lipids, to alleviate cellular stresses. In addition, during the fasting state, autophagy acts as a “metabolic rescuing” pathway to degrade non‐essential proteins, providing free amino acids for the synthesis of essential proteins. Conversely, when nutrients are abundant postprandially, insulin potently suppresses this “self‐eating” process in cells. While studies have demonstrated the important role of insulin signaling on the autophagic pathway in peripheral tissues, whether insulin signaling contributes to the regulation of the autophagic pathway in the central nervous system remains elusive. Here, we investigate the role of insulin stimulation on autophagy in mouse astrocytes, the most abundant glial cells in the brain. We show that following insulin stimulation (100 nM, 6 h), the transcription of the critical genes involved in autophagic pathway, including p62, Ulk1/2, several Atg genes, are all dramatically decreased. Consistently, the protein levels of p62, Ulk1/2, as well as LC3, are all reduced following insulin stimulation, indicating suppression of the autophagic process. The effect of insulin signaling on the transcriptional regulation is potent, since 1 nM of insulin is sufficient to suppress many of the critical autophagic genes. To confirm whether insulin suppresses cellular autophagy in astrocytes through insulin receptors, we assessed the expression of autophagic genes following insulin stimulation in both wild‐type (WT) and insulin receptor knockout (IRKO) astrocytes. Compared with WT astrocytes, insulin partially suppresses the expression of autophagic genes in IRKO astrocytes, indicating insulin also can signal through the IGF‐1 receptor in astrocytes. In summary, insulin signaling potently suppresses the autophagic process in astrocytes. This connection between autophagy gene transcription and insulin levels may be an important mechanism for the CNS‐related complications in diabetic patients.
Osmosensing by cytoplasmic biomolecular condensates of antiviral MxA GTPase
Authors: Sehgal Pravin B.
Published: May 2022

Cellular and tissue‐level edema is a common feature of acute viral infections, including covid‐19. However, there is little understanding of the effects of tissue edema on antiviral effector mechanisms. In as much as defects in IFN‐α function (such as due to presence of autoantibodies) are now implicated in enhanced severity of covid‐19 disease, we investigated how hypoosmolarity and cellular edema might affect the antiviral activity of IFN‐α‐induced human MxA GTPase, a major antiviral effector against several RNA and DNA viruses. We had previously discovered that cytoplasmic human MxA existed in the cytoplasm in phase‐separated membraneless biomolecular condensates with a gel‐like internal consistency. The vesicular stomatitis virus (VSV) nucleocapsid (N) protein associated with such MxA condensates in cells exhibiting an antiviral phenotype. Unexpectedly, MxA condensates displayed rapid and reversible osmosensing properties. Condensates of both IFN‐α‐induced endogenous MxA and of exogenous GFP‐MxA expressed in human A549 lung and Huh7 hepatoma cells rapidly disassembled within 1‐2 min when cells were exposed to hypotonic buffer (approx. 50 mOsm), and rapidly reassembled into new structures within 1‐2 min of shifting of cells to isotonic culture medium (approx. 330 mOsm). GFP‐MxA condensates in cells continuously exposed to culture medium in the range one‐fourth, one‐third or one‐half isotonicity first rapidly disassembled within 1‐2 min, and then, in most cells. spontaneously reassembled 7‐15 min later into new structures. The data are consistent with cytoplasmic “crowding” as the mechanism driving MxA condensate formation. In terms of antiviral function, one 5 min cycle of disassembly and reassembly of GFP‐MxA condensates at 1 or 2 hr after VSV infection did not affect MxA antiviral activity. Additionally, continuous exposure of GFP‐MxA expressing A549 lung cells to culture medium at one‐fourth or one‐third tonicity (thus triggering a cycle of disassembly and slower spontaneous reassembly in the continued presence of hypotonic culture medium) together with a VSV challenge, left antiviral activity largely unaffected. Such cultures showed an increase in number of cells with VSV N colocalized with the reassembled GFP‐MxA condensates. The data reveal a novel property of MxA biomolecular condensates ‐ rapid and reversible osmosensing. Importantly, antiviral activity was largely unaffected by tonicity‐driven disassembly/reassembly mimicking conditions of cellular and tissue‐level edema.
Implication of GDF15 in the Context of a Renal Adaptation to a Low Potassium Diet
Authors: Lasaad Samia , Walter Christine , Rafael Chloé , Morla Luciana , Crambert Gilles , Cheval Lydie
Published: May 2022

Introduction

In the days of hunter‐gatherers, the diet was rich in potassium (K+) and low in salt (NaCl) unlike the current modern western diet, in westernized population, which is low in K+ and rich in NaCl. In the long term, a low‐K+ diet may contribute to the development of diseases such as hypertension or acidosis. The kidney is the key organ in maintaining the electrolyte balance in our body. Indeed, its role is to adjust by different mechanisms the excretion of electrolytes to our daily intake. These mechanisms involve modifications of ion transporters expression and activity, cellular organization and composition of the renal epithelium (proliferation, trans‐differentiation). To face K+ restriction, the kidney adapts its functions to retain K+ by increasing the number of type‐A intercalated cells (ICA). Few years ago, a transcriptomic analysis performed by our laboratory revealed that a growth factor, GDF15, was strongly upregulated in renal collecting ducts of K+ depleted animals. Therefore, we hypothesize that GDF15 may be implicated in the renal adaptation to a low‐K+ diet, in part by promoting ICA proliferation.

Material and Methods

C57BL6J mice wild type or knockout for the Gdf15 gene (GDF15‐KO) were used. They were placed under a control or low‐K+ diet for 4 days for 1/ GDF15 expression measurement by RT‐qPCR in kidney or ELISA in urine and plasma 2/ metabolic analysis (urine excretion, muscle mass and K+content) and 3/ assessment of the number of ICA by immunohistochemistry on microdissected distal tubule.

Results

An increased expression of GDF15 in renal collecting duct, urine and plasma was induced in response to a low‐K+ diet. We showed that under K+ depletion GDF15‐KO mice were unable to induce an increase in the number of ICA, showing a role of this growth factor in the cell plasticity of the collecting duct. Compared to WT mice, GDF15‐KO mice exhibit a delayed renal adaptation to a K+ restriction reflected by a delayed diminution of urinary K+ excretion. This defect induces hypokalemia in GDF15‐KO mice unlike WT mice which remain normokalemic. Interestingly, we have evidence that GDF15 contributes also to the release of intracellular K+ stores in muscles, indicating that this growth factor controls both external and internal K+ balance

Conclusion

Altogether, these results demonstrate that GDF15 is a key factor in the control of the K+ balance.
Study of the Involvement of Mitochondria in Dent’s Disease Using a Mice Model
Authors: de Combiens Elise , Sakhi Imène , Frachon Nadia , Bignon Yohan , Lourdel Stéphane
Published: May 2022

Dent's disease is a rare renal tubular disorder characterized by low‐molecular‐weight‐proteinuria, hypercalciuria with nephrocalcinosis or nephrolithiasis, and progressive renal failure. It is frequently associated with inactivating mutations of the CLCN5gene coding for the 2Cl‐/H+ exchanger ClC‐5. In the kidney, ClC‐5 is mainly expressed in early endosomes of proximal tubules (PT) cells where it optimizes the function of the vacuolar H+‐ATPase to ensure proper endocytosis of low‐molecular‐weight‐proteins.

To better understand the cellular mechanisms involved in Dent’s disease, we have generated a novel Knock‐In mouse model carrying the ClC‐5 N340K pathogenic mutation. We have previously demonstrated that these mice display similar clinical features to those observed in patients with Dent’s disease. Interestingly, we observed an increase of oxidative stress in their kidney, as evidenced by higher expression of the NADPH oxidase 4, increased H2O2production, and ROS‐related total protein damage in PT cells.

Such cellular stress may arise from dysfunctional mitochondria in PT. Here, we therefore investigated the aspect of these organelles in PT cells of WT and N340K 3 month old mice using transmission electron microscopy. Compared to WT mice, mitochondria from mice carrying the N340K mutation exhibited morphologic alterations such as increased roundness, indicating that their function may be altered. Furthermore, to identify potential markers whose urinary excretion could be associated with the progression of Dent’s disease, using mass spectrometry, we performed metabolomic analyses of urine form WT and transgenic mice aged of 4, 7 and 10 months. We demonstrated changes in the excretion of metabolites that are crucial of mitochondrial metabolism (tryptophan, quinolinic acid, malate, oxaloadipate, linolenic acid and butyric acid), suggesting that the NAD+ synthesis pathway, the citric acid cycle, and beta oxidation are affected.

The present study shed more light on the pathophysiology of Dent’s disease by suggesting that a mitochondrial dysfunction is involved in this disorder.
Demystifying Cardiac Iron Deficiency in End‐stage Heart Failure
Authors: Zhang Hao , Jamieson Kristi L. , Grenier Justin , Nikhanj Anish , Tang Jack , Wang Faqi , Wang Shaohua , Seidman Jonathan , Seidman Christine , Thompson Richard , Seubert John , Oudit Gavin
Published: May 2022

Background

Cardiac iron depletion (CID) in heart failure (HF) remains largely unexplored regarding its prevalence, pathogenesis, and impact on the failing hearts. Hypotheses: We anticipate that iron‐deficient failing hearts will demonstrate defective iron regulatory pathways, whereby the HF progression is markedly aggravated by the mitochondrial redox imbalance.

Purpose

We aim to establish CID’s defining criterion, recapitulate its pathophysiological role, and evaluate the applicability of monitoring it non‐invasively in HF.

Methods

Biventricular myocardial iron levels were measured in both failing (n=138) and non‐failing control (NFC, n=46) explanted human hearts. Clinical phenotyping was complemented with comprehensive assessment of myocardial remodeling and mitochondrial functional profiles, including metabolism, respiration, and oxidative stress. Heart iron status was further investigated by cardiac magnetic resonance (CMR) imaging.

Results

Tissue iron content in the left ventricle (LV) was significantly lower in HF versus NFC [121.4 (88.1‐150.3) vs. 137.4 (109.2‐165.9) μg/g dry weight], which was absent in the right ventricle (RV). With a priori cutoff of 86.1 μg/g d.w. in LV, we identified 23% of HF patients with CID (HF‐CID) associated with higher NYHA class and worsened LV function. Respiratory chain and Krebs cycle enzymatic activities were suppressed and strongly correlated with depleted iron stores in HF‐CID hearts. Defenses against oxidative stress were severely impaired in association with worsened adverse remodeling in iron‐deficient hearts. Mechanistically, iron uptake pathways were impeded in HF‐CID, while transmembrane fraction of ferroportin positively correlated with CID. CMR with T2* effectively captured myocardial iron levels in failing hearts.

Conclusions

CID is highly prevalent in advanced human HF and exacerbates pathological remodeling in HF driven primarily by dysfunctional mitochondria and increased oxidative stress in the LV. CMR demonstrates clinical potential to non‐invasively monitor CID.
Blockade of Stim‐activated TRPC‐ORAI Channels Prevents Vascular Remodeling and Pulmonary Hypertension Induced by Chronic Intermittent Hypoxia
Authors: Castillo‐Galan Sebastian , Riquelme Barbara , Iturriaga Rodrigo
Published: May 2022

Obstructive sleep apnea (OSA), a sleep breathing disorder featured by chronic intermittent hypoxia (CIH), is associate with mild pulmonary hypertension (PH). Rats exposed to CIH developed pulmonary vascular remodeling and PH, but the pathogenic mechanisms are not well known. In addition, CIH overexpressed Stim‐activated TRPC‐ORAI channels (STOC) in the lung, which paralleled the increase of right systolic ventricle pressure (RVSP) and pulmonary vascular remodeling. Accordingly, we tested weater a treatment with the STOC bloker 2‐Aminoethyl diphenylborinate (2‐APB), started from day 14 days to exposure to CIH may reduce vascular remodelling and PH. Male Sprague‐Dawley rats (~200g) were exposed CIH (5% O2, 12 times/h for 8h). At 14 days, osmotic pumps containing 2‐APB (10 mg/kg/day, n=7) or its vehicle (n=7) were implanted, and rats were keep in CIH for 2 more weeks. At the end of CIH exposure, RVSP was measured in urethane‐chloralose anesthetized rats. Animals were euthanized and lungs were removed to determine vascular remodeling and mRNA levels of the STOC forming subunits TRPC1, TRPC4, TRPC6, ORAI 1 and ORAI 2 by qPCR. CIH‐treated rats were compared with aged‐matched normoxic controls. 2‐APB reduced the increase of RVSP (24.7 ± 1.6 vs. 36.5 ± 1.1 mmHg, 2‐APB vs vehicle, respectively, p < 0.05) and the medial layer thickness of arteries (50‐300 µm) (43.4 ± 1.1 vs. 66.5 ± 2.9 %, 2‐APB vs vehicle, respectively, p < 0.05). In addition, 2‐APB prevented the increase of α‐actin‐ir (8.8 ± 0.4 vs. 14.3 ± 0.6 pixels/μm2, 2‐APB and vehicle respectively, p < 0.05), and to KI67 positive cells (3.9 ± 0.8 vs. 6.3 ± 1.1 %, 2‐APB and vehicle, respectively, p < 0.05). In addition, 2‐APB treatment reverted the increased mRNA levels of TRPC1, TRPC4 and TRPC6, but induced an overexpression of ORAI 1, while ORAI 2 remained unchanged. The present results show that 2‐APB treatment prevented the vascular pulmonary alterations induced by CIH, suggesting that the blockade of STOC may potentially be used to treat the pulmonary hypertension induced by OSA.
Calcineurin inhibition causes persistent hypertension through hypothalamic NMDA receptor‐dependent sympathetic outflow
Authors: Zhou Jing‐Jing , Shao Jian‐Ying , Chen Shao‐Rui , Pan Hui‐Lin
Published: May 2022

Calcineurin is a calcium/calmodulin‐dependent serine/threonine protein phosphatase, and hypertension is a major side effect of clinically used calcineurin inhibitors. Although increased sympathetic nerve discharges are associated with calcineurin inhibitor‐induced hypertension, the sources of sympathetic outflow are unknown. In this study, we determined the role of the paraventricular nuclear (PVN) of the hypothalamus in calcineurin inhibitor‐induced hypertension. Immunocytochemistry labeling showed that calcineurin was expressed on spinally projecting PVN neurons. Radiotelemetry recordings showed that systemic administration of tacrolimus (FK506), a specific calcineurin inhibitor, for 14 days caused a gradual and significant increase in arterial blood pressure in rats, which lasted at least another 7 days after discontinuing FK506 treatment. FK506 significantly decreased calcineurin activity in the PVN and increased the protein level of GluN1, the NMDA receptor obligatory subunit, and serine/threonine phosphorylation levels of GluN1 in the PVN synaptosomes. Electrophysiological recordings revealed that the spontaneous firing rate, frequency of mEPSCs, and amplitude of puff NMDA‐elicited currents in spinally projecting PVN neurons were significant greater in FK506‐treated rats than in vehicle‐treated rats. Remarkably, treatment with the NMDA receptor antagonist AP5 normalized the stimulatory effects of FK506 on PVN neurons of FK506‐treated rats. Furthermore, microinjection of AP5 into the PVN decreased renal sympathetic nerve discharges and blood pressure increased in FK506‐treated rats. Systemic administration of memantine, a clinically used NMDA receptor antagonist, also significantly lowered the high blood pressure in FK506‐treated rats. Collectively, our findings indicate that calcineurin inhibitor‐induced persistent hypertension results from augmented sympathetic output through increased NMDA receptor phosphorylation and activity in the PVN.
Inhibition of PRMT5 Disrupts Cell Cycle Progression and DNA Damage Signaling, Revealing a Potential Novel Combination Therapy for Pancreatic Cancer
Authors: He Lishu , Urrutia Guillermo , Lomberk Gwen
Published: May 2022

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and remains a therapeutic challenge. There is an urgent need for novel therapeutic strategies for PDAC, and the targeting of pathways highly relevant to its pathobiology is critical. PDAC cells often possess defects in DNA damage repair (DDR) pathways that are not present in healthy cells, which can be exploited for therapeutic strategies. Protein arginine methyltransferase 5 (PRMT5) is a type II histone arginine methyltransferase involved in various cellular processes, such as cell cycle progression and DDR. In PDAC tumors, PRMT5 expression is elevated and predicts shorter overall survival and disease‐free patient survival than those with lower PRMT5 expression. Here, we examined the effect of PRMT5 inhibition on PDAC in vitro, the impact of its inhibition on cell cycle and DDR, as well as potential synergy between PRMT5 inhibition and DNA damage‐inducing ionizing radiation (IR) as a novel therapeutic strategy for PDAC. Treatment of PDAC cells withthe PRMT5 inhibitor (PRMT5i), EPZ015938, resulted in a dose‐dependent decrease of cell growth by time‐lapse live‐cell imaging and clonogenic survival assays. Flow cytometry‐based cell cycle analysis showed that PDAC cells treated with EPZ015938 had increased numbers in G2/M, suggesting that PRMT5 inhibition triggers a G2/M arrest. Using a phospho‐protein array of cell cycle control proteins, we found higher levels of WEE1, P‐S216‐CDC25C, and P‐Y15‐CDC2, supporting G2/M arrest following treatment with EPZ015938. The phospho‐protein array also revealed an increase in P‐S15‐TP53, a signal of DNA damage mediated by the DNA damage sensors, ATM/ATR, along with a decrease in ATM. We confirmed the ATM downregulation at the transcript level by quantitative RT‐PCR. Pathway‐specific qPCR array analysis displayed differential expression of DDR genes related to ATM and G2/M DNA damage checkpoint regulation, further implicating a mechanistic role for these pathways in response to PRMT5i. The downregulation of ATM combined with the presence of G2/M arrest and signals of DNA damage led us to examine the related ATR pathway. In protein lysates from PDAC cells treated with PRMT5i followed by western blot, we found increased phosphorylation of ATR at S1981 and H2A.X at S139. This suggests that PRMT5 inhibition activates DDR in PDAC cells via ATR. To harness the potential vulnerability presented by PRMT5i‐mediated G2/M arrest, we combined PRMT5i treatment with ionizing radiation (IR) exposure to find that the combination of EPZ015938 and IR displayed a greater reduction in PDAC cell growth in vitro over individual treatments. In conclusion, we find that PRMT5 is a critical player in DNA damage via modulation of the ATM/ATR pathways, and combined PRMT5i and IR have beneficial anti‐tumor effects in vitro, revealing an exciting avenue for further exploration as a potential new therapeutic strategy in PDAC.
Decoding Medication Tradenames; an innovative Method for Top 200 medications in Didactic & Experiential Courses
Authors: Elsobky Yasmin , Mente Welly , Kreys Eugene , Mohamed Islam
Published: May 2022

Objective

To investigate whether decoding of top 200 medication tradenames can result in improving pharmacy students’ performance in IPPE‐didactic & experiential courses. For ex: Macrobid®; provides an elegant explanation of (MACRO) size crystal form of (Macrodantin) designed for slower absorption & less excretion, hence, used twice daily (BID).

Methods

An educational intervention of decoded tradenames (explanation of techniques used in top 200 tradenames) was implemented using Quizlet & summary‐tables before the summative final exam in a first‐year didactic IPPE course. Aggregate de‐identified students’ scores were used for blinded‐paired statistical‐analyses. A Likert‐scale‐surveys was used for evaluating students’ quality of learning experience after both the IPPE‐didactic and experiential rotations courses.

Results

In aggregate analyses from Fall 2019‐2020; paired data analyses using Wilcoxon test showed a statistically significant increase in average students’ performance from 70.6% +/‐ 18.9% in formative to 80.5% +/‐ 23.5% in summative assessments after the intervention in control questions involving tradenames without rational explanation (p‐value<0.001). Whereas, in questions involving tradenames that have rational explanations, students’ performance increased from an average of 58.0%+/‐28.3% before the intervention, to 75.9%+/‐22.2% afterwards, which represents a net significant difference of 8.1%+/‐39.8% between question groups (p‐value0.009) (n= 169 students). In addition, at least 80% of students reported that decoding tradenames can help them to retain key medications features & connect it to generic names during didactic course, hence, provide better medication recommendations, & make less potential medication errors during their experiential rotation (n=72/94 students).

Conclusions

Supplementing pharmacy curricula with the art & science of formulating medication tradenames can improve students’ academic performance in didactic courses. Furthermore, it can also improve the resulting quality of healthcare services future pharmacists provide during their experiential education.