bluuebunny/crossref_metadata_2025_split huggingface dataset:
MYTHO: A novel regulator of autophagy and skeletal muscle health
Authors: Leduc‐Gaudet Jean‐Philippe , Franco‐Romero Anaïs , Broering Felipe E. , Moamer Alaa , Cefis Marina , Chaffer Tomer J. , Dulac Maude , Marcangeli Vincent , Sedraoui Sami , Shams Anwar , Mayaki Dominique , Huck Laurent , Petrof Basil , Lochmuller Hanns , Sandri Marco , Hussain Sabah N. , Gouspillou Gilles 
Published: May 2022

RationaleThe loss of skeletal muscle function can have dramatic health consequence. Skeletal muscle dysfunction are found in a wide range of clinical conditions, including cancer cachexia, starvation, sepsis, and aging. Autophagy is an essential catabolic process that remove damaged cytosolic components. Accumulating evidence indicates that insufficient or excessive autophagy can contribute to the development of skeletal muscle atrophy. We recently identified a novel FoxO‐dependent gene, which we named MYTHO, with the potential to regulate autophagy in skeletal muscles. However, to date, the roles that MYTHO plays in skeletal muscles remain unknown. In this study, we evaluated the functional importance of MYTHO in skeletal muscle structure and function.MethodsTo study the functional importance of MYTHO in skeletal muscle mass and function, we used a combination of genetic strategies in mice to delete or overexpress MYTHO.ResultsMYTHO expression is induced in various muscle wasting conditions, including starvation, denervation, cancer cachexia, and sepsis. Using different genetics approaches, we confirmed that MYTHO is an important regulator of autophagy in skeletal muscle. Importantly, we also found that MYTHO knockdown in skeletal muscles caused various myopathic features, including muscle weakness, accumulation of tubular aggregates, a shift in myofiber type composition and nuclei mispositioning in myofibers. Using animals with muscle specific Atg7 deletion (a model of autophagy inactivation), we demonstrated that the pathological features associated with MYTHO knockdown are independent of its role in regulating autophagy.ConclusionWe conclude that MYTHO is a central player in regulating skeletal muscle mass and integrity.
Renal Venous Clamping Exacerbates Outer‐Medullary Red Blood Cell Congestion during Ischemia in Male and Female WKY Rats
Authors: Johnson Chloe , McLarnon Sarah , Sun Jingping , O'Connor Paul 
Published: May 2022

Despite receiving approximately 20% of cardiac output, the kidneys are highly susceptible to ischemic injury, which is most prominent in the renal outer medulla. Previous studies have suggested that vascular congestion may explain the susceptibility of this region to injury. Further, increased injury has been reported in models where the renal vein is occluded compared to the renal artery. The goal of the current study was to determine whether renal arterial versus venous clamping would alter the severity of red blood cell congestion in the outer medulla. We hypothesized that venous clamping would result in greater vascular congestion than arterial clamping due to continued inflow of arterial blood.Ischemia reperfusion (IR) surgeries were performed on male and female WKY rats (10‐14 weeks). For each animal, the renal artery for one kidney and renal vein for the other kidney were clamped for a period 15 (n=7 male, 6 female) or 45 (n= 6 male, 5 female) minutes. The kidneys (right or left) which received arterial or venous clamping were rotated for each animal. Following ischemia, the kidneys were excised while the clamps remained on the vessels, without reperfusion. Vascular congestion of the outer medulla was scored blinded on a scale of 0 (0% congestion) to 5 (100% congestion) in trichrome stained kidneys sections.We found that there was significantly greater congestion of red blood cells in both the vasa recta and outer‐medullary plexus with venous clamping when compared to arterial clamping for both 15 minutes (p=0.0008, p<0.0001, respectively) (Fig. A) and 45 minutes (p=0.0001, p<0.0001, respectively) (Fig. B). There was no significant effect of sex observed on degree of vascular congestion regardless of the location or duration of the clamp.Our data indicates that red blood cell congestion is exacerbated by renal venous occlusion, which may explain the increased tubular injury that has been observed in this model. Therefore, future studies will investigate injury following recovery from renal artery versus vein clamping. Understanding the cause(s) of vascular congestion is essential for the development of clinically effective treatment options for acute kidney injury. Novel therapeutic approaches that prevent or reverse red blood cell congestion of the renal medulla may be effective in limiting ischemic acute kidney injury.
Synthesis of the transmembrane domain of the spike protein from SARS‐CoV‐2 using solid phase peptide synthesis and determination of its oligomerization state
Authors: Grimes Loyal T. , Reichart Timothy M. 
Published: May 2022

As of today, nearly six hundred thousand lives have been lost to the disease COVID‐19 in the United States alone. This highly contagious disease has a proposed R0 value of 5.7, meaning that for every person infected, they transmit the virus to about 5 other people. This has caused researchers to expeditiously study Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2). Due to the state of the current pandemic, the distribution of effective vaccination and antiviral medication is paramount to global health. The Spike (S) protein in SARS‐CoV‐2 is largely responsible for the reproduction of this virus. The S protein consists of a receptor binding domain (RBD), which the virus uses to bind to angiotensin‐converting enzyme 2 (ACE2) receptors of host cells (REF) as well as a stem like structure binding to the membrane. The fusion of the viral and cell membranes allow for the release of the viral genome. It is known that the Spike protein has a trimerized oligomerization state, which is believed to be necessary to its function. The goal of this project is then to synthesize, characterize, and analyze the function of the transmembrane domain (TM) of the S protein in SARS‐CoV‐2. The native sequence of this peptide is the following:WYIWLGFIAGLIAIVMVTIMLIn order to determine the oligomerization state of the TM Domain of the S protein, that being how many times the peptide may repeat or come together with identical units of itself. By conducting a fluorescence‐based oligomerization assay this may be determined. This method relies upon having the native sequence of the domain separate from the entire protein to determine the specific effect of oligomerization state on the TM domain. The most practical method to synthesize the TM domain of the S protein is through Solid Phase Peptide Synthesis (SPPS). SPPS is a process in which peptides are made by linking amino acids, the monomers of proteins, one at a time until the full sequence is achieved. These peptide chains will additionally need to be purified and quantified through high‐performance liquid chromatography (HPLC). The synthesized peptides will be analyzed using liquid chromatography‐mass spectrometry (LCMS) to determine the prevalence and mass of the synthesized peptides. If the masses correlate to the same masses found in the S protein of SARS‐CoV‐2, then the correct peptides will have been synthesized correctly. The continued investigation of the S protein can lead to the discovery of small peptides capable of inhibiting key processes to the binding mechanism of SARS‐CoV‐211‐13. The function of the S protein is believed to only present when the peptides from trimers. Therefore, the analysis of their oligomerization states, that is how well trimerization can occur, will be investigated by synthesizing versions of the peptide that fluoresce when excited using dyes such as nitrobenzodiazole (NBD) and TAMRA in the fluorescence assay.
Membrane phosphoinositides and renal epithelial cell polarity determination in the Xenopus pronephros in vivo
Authors: Schena Giorgia , Rajendran Vanathy , Khokha Mustafa , Caplan Michael 
Published: May 2022

BackgroundThough they are only minor components of cell membranes, phosphoinositide lipids (PIs) participate in numerous signaling processes and in membrane identity determination. Many studies of cultured MDCK renal epithelial cells found that PIs are distributed with polarity among plasma membrane (PM) domains, and that their polarized distributions are required for the delivery of distinct populations of apical and basolateral membrane proteins. The extent to which PIs drive these processes in actual renal epithelial cells in vivo has never been examined. We investigate the distribution of PIs in vivo in the pronephros of Xenopus laevis tadpoles using MCherry‐tagged Pleckstrin Homology (PH) domains that selectively bind different PIs (PH‐AKT, PH‐PLCD1, which bind to (PI(3,4,5,)P3 and PI(4,5)P2 respectively) with the goal of assessing whether and how PI localizations affect cell polarity determination and the trafficking of proteins to their sites of ultimate functional residence in renal epithelial cells in situ.MethodsmRNA encoding MCherry‐PH‐AKT or MCherry‐PH‐PLCD1 was injected into fertilized oocytes and their distributions were assessed in the developed pronephros at stage NF45 via fluorescence microscopy. Knockdown (KD) of PTEN, a lipid 3’ phosphatase that regulates membrane PI composition, was achieved via injection of targeted morpholinos and confirmed by western blotting. The effects of PTEN KD on MCherry‐PH‐AKT distribution were assessed by fluorescence microscopy.ResultsIn MDCK cells PH‐AKT and PH‐PLCD1 localize to the basolateral and apical PMs, respectively. Their distributions are quite different in the pronephros, with both sensors localizing apically in the proximal portion of the tubule and basolaterally in the distal part. Interestingly, PTEN localization, at least in proximal tubules, is apical and not different than previously reported in cell models. Finally, PH‐AKT staining dramatically re‐distributes to the lateral domain of renal cells upon PTEN KD but this treatment does not alter the localization of protein markers of epithelial PM polarity.ConclusionsThese studies constitute the first effort to assess the role of PIs in establishing PM polarity in renal epithelial cells in vivo. Our data reveal important differences from observations previously made with in vitro systems. These findings highlight the need to explore the processes that produce renal epithelial cell polarity in vivo and in the context of intact renal tubules.
Aquaporin 1 mediates microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary hypertension
Authors: Yun Xin , Philip Nicolas M. , Jiang Haiyang , Smith Zion , Huetsch John , Mahendra Damarla , Suresh Karthik , Shimoda Larissa 
Published: May 2022

Pulmonary arterial hypertension (PAH) is a devastating disease, with increased pulmonary arterial pressure leading to right heart failure. The mechanisms underlying PAH are unclear, but increased pulmonary arterial smooth muscle (PASMC) and microvascular endothelial cell (MVEC) migration and proliferation leads to vaso‐occlusive lesions and vascular remodeling observed in PAH. Aquaporins regulate water transport and are expressed in many cells. We previously identified that hypoxia upregulates the expression of aquaporin 1 (AQP1) in PASMC and elevated AQP1 was required for hypoxia‐induced migration and proliferation. In this study, we wanted to elucidate whether the same is true in MVECs, and used the SU5416 plus hypoxia (SuHx) rat model, a robust model of PAH, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in MVECs and contributed to a hyperproliferative/hypermigratory phenotype.Rats were injected with the vascular endothelial growth factor receptor inhibitor, SU5416, combined with hypoxia (3 wk at 10% O2) followed by 2 wk return to normoxia. Using MVECs isolated from the normoxia (N‐MVEC) or SuHx (SuHx‐MVEC) rats, we found that AQP1 protein levels were increased in SuHx‐MVECs and associated with upregulation of AQP1 mRNA levels, even in the absence of sustained hypoxia exposure. Silencing of AQP1 reduced migration and proliferation observed in SuHx‐MVEC, whereas augmenting AQP1 levels in N‐MVECs using forced expression with an adenoviral construct containing wild‐type AQP1 (AdAQP1) was sufficient to increase cell migration (transwell assay) and proliferation (BrdU incorporation) compared to control cells infected with green fluorescent protein (AdGFP). Since we formerly reported the C‐terminal tail of AQP1 contains putative calcium and protein biding sites and is required for enhanced PASMC migration and proliferation, we tested the role of the AQP1 C‐terminal tail in N‐MVEC. Cells were treated with AdGFP, AdAQP1 or adenovirus containing AQP1 lacking the entire cytoplasmic C‐terminal tail (AdAQP1CT). In contrast to the increase in migration and proliferation induced by AdAQP1, infection with AdAQP1CT did not alter N‐MVEC migration and proliferation.Our results suggest a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in microvascular endothelial cells from a rodent model that reflects many of the features of human PAH. Interestingly, the AQP1 C‐terminal tail is required for regulating MVEC function, similar to our previous findings in PASMC.
Single‐Cell Sequencing Reveals Distinct Expression Patterns of the Integrated Stress Response (ISR) Pathway Regulating Cell Survival in Secretory Cells in Colon of Hnrnp I Knockout Mice
Authors: Xu Guanying B. , Pan Yuan‐Xiang , Mei Wenyan , Chen Hong 
Published: May 2022

ObjectivesHeterogeneous nuclear ribonucleoprotein I (Hnrnp I) is an RNA‐binding protein functions during pre‐mRNA splicing and regulation of alternative splicing events. Previously we found that ablation of Hnrnp I activates NF‐κB signaling in intestinal epithelial cells (IECs), resulting in increased nuclear translocation of P65 (NF‐κB subunit) and upregulation of proinflammatory cytokines and chemokines. Integrated stress response (ISR) signaling was linked to activation of canonical NF‐κB pathway. Therefore, in this study, we aimed to investigate ISR in the colon of Hnrnp I knockout mice. We hypothesize that cellular stress from Hnrnp I knockout triggers ISR, consequently affects the cellular dynamics among secretory cell types on colon epithelium (enteroendocrine, Paneth and goblet cells).MethodsTotal RNA was isolated from total colon cell preparations from WT and KO mice. Single‐cell RNAseq libraries (biological replicates, n=3) were prepared using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters, a computational cell calling algorithm was used to identify cell type using two published annotated mouse cell datasets. Pathway analysis was performed for secretary cell clusters including enteroendocrine cells (EECs), Paneth cells, and goblet cellsResultsComparing to WT, number of EECs in colon was significantly increased in KO mice. Total mRNA level of Atf4, the principal regulator of ISR, was significantly increased in colon secretory cells in KO mice. We also examined mRNA expression of ATF4 target genes in secretory cells. Specifically, gene encoding the regulator of apoptosis and autophagy, members of Bcl2 family, Bnip3, was significantly increased in secretory cells in KO mice, while Mlx and Gabarapl2 (Atg8), both are involved in apoptosis and autophagy signaling pathways, have a trend of increase in secretory cells in KO mice, all comparing to WT.ConclusionsCellular stress from Hnrnp I knockout activated ISR in IECs, specially in those secretory cell types – EECs, Paneth, and goblet cells. The activation of ISR led to both autophagy and apoptosis signaling in these cells through the upregulation of Bnip3, Mlxand Gabarapl2. Taken together, our results suggest that in response to the cellular stresses from the depletion of Hnrnp I in IECs, ISR activation in colonic secretory cells, stimulating apoptosis and autophagy pathway signaling, particularly in EECs, resulting in the proliferation of EECs in KO. The overall manipulation by ISR signaling in the colon secretary cells may present a critical early response to modulations in colon, potentially leading to perturbations to homeostasis among host colon epithelia, colon residential immunity, and gut microbiota.
Assessing Contractility of 3D iPSC‐derived Muscle Models for Safety and Discovery Using a Novel, High‐throughput, and Label‐free Instrumentation Platform
Authors: Geisse Nicholas , Berry Bonnie J. , Gray Kevin , Moerk Travis , Person Jason , Luttrell Shawn M. , Macadangdang Jesse 
Published: May 2022

Stem cell models hold great promise for improving the predictive power of preclinical in vitro assays for new therapies, drug discovery, basic scientific research, and disease modeling. Complex 3D platforms, such as Engineered Muscle Tissues (EMTs) fabricated from primary or iPSC‐derived cells, can directly measure tissue contractility, which is challenging in conventional 2D platforms where cells are rigidly attached to a surface. However, traditional methods to fabricate EMTs demand extensive bioengineering expertise, and measuring contractility often involves laborious, serial, and low‐throughput optical measurements.Here, we report on the design, fabrication, and validation of a novel 3D EMT platform that uses 1) facile and scalable bioengineering approaches to generate tissues from a variety of cell sources, and 2) a label‐free parallel measurement technique. Our tissue casting approach has a success rate of >96% (n > 100) and produces consistently‐sized constructs with a standard deviation of +/‐ 9% across 6 experiments. Tissue casting, media changes, and drug dosing are also highly amenable to automation.The substrate features an embedded magnet; as tissues contract, the magnet’s displacement is quantitatively detected in a highly‐parallel manner using specialized sensors. We detected 24 contractions simultaneously with a measurement rate of 100Hz, which is suitable for measuring various aspects of contractility such as upstroke velocity, decay time, and fatigability. We demonstrated that the signal voltage changes are linear with respect to EMT contraction.We will present data showing acute effects of drugs measured minutes after EMT dosing and chronic effects of structural cardiotoxicants like doxorubicin, sunitinib, and BMS‐986094 on EMTs. All chronically‐dosed tissues showed statistically significant dose‐dependent reduction in twitch frequency over a multi‐day time course (p <0.05). We will also show that our platform can be used to generate physiologically‐relevant skeletal muscle constructs and achieve tetanic responses upon stimulation.In addition to modeling healthy tissues, our platform can also be used to study disease models. We are currently developing patient‐specific Duchenne Muscular Dystrophy models for the development of personalized gene therapies. EMTs can be made from cells sourced from patients and used to test whether a new therapy will improve or recover functional contraction.We have designed a novel system that can leverage the complexity of 3D cellular models in a scalable format and can be tailored for specific applications, including personalized medicine or disease modeling. The Mantarray platform will provide a stand‐alone tool capable of screening significant numbers of compounds for the rapid safety evaluation of drug candidates thereby accelerating drug discovery and development.
Renal‐Tubular SGK1 is Involved in Blood Pressure Circadian Rhythm
Authors: Al‐Qusairi Lama , Debonneville Anne , Stifanelli Matteo , Basquin Denis , Staub Olivier 
Published: May 2022

As many physiological functions, blood pressure (BP) follows a circadian pattern that rises during the active and decreases during the inactive phase of the day. BP rise at the beginning of active phase (known in human as the “morning surge”) coincides with the increase in circulating glucocorticoids and aldosterone. Serum‐ and glucocorticoid‐induced kinase (SGK1), a clock‐controlled and aldosterone‐induced gene, plays a role in BP regulation in human and animal models. However, a role of SGK1 in the BP circadian regulation has not been demonstrated.Using telemetry, we analyzed BP in the inducible renal‐tubule specific Sgk1Pax8/LC1 model under basal and high‐potassium diet (HKD; 5%K+). Our data show that, under basal conditions, renal SGK1 plays no role in BP regulation. After one week of HKD and despite largely unaffected mean arterial BP (MAP), Sgk1Pax8/LC1 mice exhibit altered day/night variations. More specifically, the increase in BP during the active phase (BP surge) was blunted in both systolic and diastolic BP. After 7 weeks of HKD, more profound defect in BP was observed in the mutants as they exhibit decreased MAP, together with a dysregulated circadian rhythmicity. The BP defect was associated with abnormal NCC and ENaC expression and processing. Altogether, our data suggest that i) The kidney plays a major role in setting the BP circadian rhythm. ii) BP surge requires the renal‐tubule SGK1.
Renal chemosensitive and mechanosensitive afferent responses are preserved in TRPV1 knockout rats
Authors: DeLalio Leon , Stocker Sean 
Published: May 2022

The transient receptor potential vanilloid type‐1 (TRPV1) channel is expressed in renal sensory nerves, and intrarenal administration of the TRPV1 agonist capsaicin increases renal afferent nerve activity (ARNA). Nonselective denervation of renal sensory nerves using high concentration capsaicin reduces arterial blood pressure (ABP) in experimental models of hypertension. Pharmacological blockade of TRPV1 using renal pelvic infusion of capsazepine attenuates ARNA responses to capsaicin, CGRP, and elevated pelvic pressure. Thus, TRPV1 channels mediate activation of renal sensory nerves.To test this hypothesis, we generated a novel TRPV1 knockout rat (TRPV1‐/‐) using CRISPR/Cas9 to produce a 26bp deletion in exon 3. Multifiber ARNA responses to chemical (bradykinin and capsaicin) and mechanical (elevated renal pelvic pressure) stimuli were compared in Inactin‐anesthetized male and female wild‐type and TRPV1‐/‐ littermates (250‐400g). There were no significant differences in baseline mean ABP between wild‐type and TRPV1‐/‐ rats (122±2 mmHg vs 122±3 mmHg, respectively; p=0.48, n=8‐10), or baseline ARNA between wild‐type and TRPV1‐/‐ rats (14±3 Hz vs 18±5 Hz, respectively; p=0.24, n=8‐10).Intrarenal artery infusion of capsaicin (0.1–10μM, 50μL per 15s) significantly increased ipsilateral ARNA in wild‐type but not TRPV1‐/‐ rats (Δ discharge 65±14 Hz vs 9±3 Hz at 10μM, respectively; p<0.05, n=7). As a second chemosensitive stimulus, intrarenal artery infusion of bradykinin (0.1–10μM, 50μL per 15s) produced significant increases in ARNA in ­both wild‐type and TRPV1‐/‐ rats that were not significantly different between groups (Δ discharge of 10μM: 52±11 Hz vs 76±21 Hz, respectively; p=0.33, n=7). Elevated renal pelvic pressure (0–20mmHg; 30s) significantly increased ARNA in both wild‐type and TRPV1‐/‐ rats; however, ARNA responses were significantly smaller in wild‐type versus TRPV1‐/‐ rats (Δ discharge with 20mmHg: 17±3 Hz versus 29±6 Hz, respectively; p=0.03, n=8‐9).The preservation of ARNA responses in TRPV1‐/‐ rats contradicts prior studies using pharmacological TRPV1 antagonists. A final set of experiments tested whether intrapelvic capsazepine infusion (200μM, 200μl per 3 min) attenuates ARNA responses to elevated pelvic pressure (20mmHg) in both wild‐type and Trpv1‐/‐ rats. Interestingly, pelvic administration of capsazepine attenuated mechanosensitive ARNA responses in both wildtype (saline: 31±3 Hz vs capsazepine: 8±1 Hz; n=3) and TRPV1‐/‐ (saline: 20±7 Hz vs capsazepine: 6±1 Hz; n=3) rats thereby suggesting a TRPV1‐independent action of capsazepine.In conclusion, TRPV1 channels are dispensable for the activation of chemo‐ and mechanosensitive renal sensory nerves.