bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ying and the Yang: Compensatory UPR Signaling Responses Observed In An In‐Vitro Model Expressing Clinical Mutant Surfactant Protein C Isoforms
Authors: Dimopoulos Thalia , Iyer Swati , Rodriguez Luis R. , Murthy Aditi , Katzen Jeremy , Beers Michael F.
Published: May 2022

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by alveolar type II (AT2) cell dysfunction. Mutations in surfactant protein C (SP‐C) are a high effect size, recognized etiological cause of IPF. The SP‐C proprotein contains an ~100 KDa Bri2 chondromodulin‐1 and prosurfactant protein C (BRICHOS) domain that acts as a molecular chaperone preventing protein aggregation. The presence of misfolded proteins in the ER can be sensed by three transmembrane sentinel proteins – activating transcription factor 6 (ATF6), protein kinase R‐like ER kinase (PERK), and inositol‐requiring enzyme 1α (IRE1α) – which trigger an intracellular signaling pathway called the unfolded protein response (UPR). Previous characterization of our in‐vitro and in‐vivo models has demonstrated that BRICHOS mutants activate all 3 arms of the UPR, activate a proinflammatory cascade, and drive AT2 cell death. In‐vivo, their expression initiates pathological fibroproliferative lung remodeling, characteristic of human IPF. Using our in‐vitro models of BRICHOS‐driven ER stress, we explore potential IPF therapeutic intervention targeting two upstream sensors of the UPR, PERK and IRE‐1.

Mouse lung epithelial (MLE‐12) cells were transfected with wildtype (WT) or BRICHOS mutant (C121G) SP‐C plasmids in media containing inhibitors of either PERK signaling or IRE‐1 RNase activity. Pathway readouts included: quantification of cell death via LDH cytotoxicity and flow cytometry, protein immunoblots to detect UPR activation and apoptotic signaling, QPCR of spliced X‐Box Binding Protein 1 (XBP1) and UPR transcriptional targets, and high‐resolution microscopy to observe altered SP‐C trafficking.

Within 48 hours of transfection of MLE‐12 cells with SP‐CC121G we observed activation of the 3 arms of the UPR, upregulated downstream targets, and initiation of cell death. Inhibition of PERK resulted in down‐regulation of PERK pathway proteins and transcriptional targets, increased SP‐C protein expression, and promoted increased XBP1 splicing. Inhibition of IRE1 RNase inhibited splicing of XBP1, reduced expression of XBP1 transcriptional targets, and increased JNK phosphorylation. Inhibition of either pathway alone did not reduce cell death, though partial reduction of cell death was noted with either small molecule targeted caspase 3/8 or necroptosis inhibition.

Our data suggests that while intervention directed at one arm of the UPR may block proinflammatory signaling or transcriptomic reprogramming, this strategy may not be sufficient to completely inhibit cell death. These findings highlight the interconnectivity and redundancy of the cell quality control machinery and the emerging role of necroptosis due to chronic UPR stress‐mediated cell death. Further characterization of this pathway is needed to improve IPF therapeutic outcomes.
Eukaryotic RNA Polymerase ⍺‐like Subunits Heterodimerize by Distinct Interaction Mechanisms
Authors: Belkevich Alana , Pascual Haleigh , Fakhouri Aula , Knutson Bruce
Published: May 2022

DNA‐dependent RNA Polymerases (Pols) are present in every living cell and even encoded by some viruses. Pols are responsible for the process of transcription, and Pols from the three domains of life are constructed of a conserved core complex that includes a dimer of α or α‐like subunits that serve as a scaffold for Pol assembly. Bacteria and archaea each encode a single Pol that transcribes all forms of RNA, while eukaryotes encode three specialized Pols (I‐III) containing one of two distinct α‐like heterodimers. One α‐like heterodimer is shared between Pols I and III, while there is a paralogous Pol II heterodimer. The α‐like subunits are clinically relevant as mutations in the Pol I/III heterodimer are associated with Treacher Collins Syndrome and 4H Leukodystrophy, while mutations in the Pol II heterodimer are associated with Primary Ovarian Insufficiency. These mutations often result in defects in Pol assembly and/or activity, although the vast majority remain uncharacterized. It is currently unclear if heterodimer formation is functionally similar in α‐like subunit orthologs and/or paralogs and how these similarities and differences can be used to study the α‐like subunit associated diseases. To examine this, we mutated several regions of the yeast and human small α‐like subunits to test their contribution to heterodimer interaction using several types of biochemical and genetic assays. Here we show that different regions serve differential roles in heterodimerization, in a polymerase‐ and species‐specific manner, with both human subunits being more sensitive to mutations. This suggests that although the subunits are evolutionary conserved, they interact differently. More broadly, these findings help explain why some disease mutations have little to no effect in yeast and possibly other model systems, and may inform us how to make better disease models in the future.
Deletion of the Vitamin D Receptor in Skeletal Muscle is Associated with Improved Glucose Tolerance and Reduced Muscle Function
Authors: Losbanos Louis , Craig Theodore , Fadra Numrah , Nair Asha , Matveyenko Aleksey , Kumar Rajiv
Published: May 2022

Skeletal muscle insulin resistance contributes to the pathogenesis of type 2 diabetes mellitus. Epidemiological studies have demonstrated that vitamin D deficiency is associated with the presence of type 2 diabetes in humans. To probe the role of the vitamin D receptor (VDR), an intracellular and nuclear protein that mediates the bioactivity of the active vitamin D ligand, 1,25‐dihydroxyvitamin D, in skeletal muscle mediated glucose disposal, we deleted the skeletal muscle VDR in adult mice. We assessed glucose disposal, insulin concentrations and sensitivity, and skeletal muscle gene expression in Vdr knockout and wild‐type Vdr mice. Following the induction of a skeletal muscle‐specific Crerecombinase with tamoxifen in adult male Vdrfl/flmice, efficient deletion of the Vdrwas demonstrated. In skeletal muscle‐specific Vdrknockout mice, basal glucose concentrations were similar to those in wild‐type Vdrmice. In Vdrknockout mice, blood glucose concentrations decreased more rapidly following the administration of intraperitoneal glucose than in control mice expressing wild‐type Vdr (Figure 1A and Figure 1B). The AUC for glucose concentrations in Vdrfl/fl.actin iCre mice treated with tamoxifen was reduced by 20% (p=0.015) compared to the AUC of blood glucose concentrations prior to tamoxifen treatment. Pre‐and post‐treatment AUC’s for blood glucose in Vdrfl/fl.actin iCre mice treated with vehicle and in Vdrfl/flmice treated with tamoxifen were unchanged (p=0.97,0.23). Plasma insulin concentrations were identical in knockout and wild‐type Vdr mice following a bolus of glucose, demonstrating that deletion of the Vdr in skeletal muscle did not influence the release of insulin in response to glucose (one‐way ANOVA, p=0.45). Insulin sensitivity was similar in Vdrknockout and wild‐type Vdrmice. Gene expression analysis of skeletal muscle mRNA derived from Vdrknockout mice and wild‐type mice demonstrated an increase in the expression of genes associated with oxidative phosphorylation, fatty acid oxidation and adipogenesis pathways that could potentially alter glucose utilization. We conclude that selective deletion of the Vdr in the skeletal muscle of adult mice results in more efficient glucose disposal than in wild‐type Vdrmice; basal and glucose‐stimulated insulin serum concentrations are identical in knock‐out and wild type VDR mice with no changes in insulin sensitivity. The data suggest that modulation of the vitamin‐D endocrine system is unlikely to improve glucose utilization.
Recovery of hyperoxia‐induced DDAH1 suppression with FXR agonist GW4064 in human pulmonary microvascular endothelial cells
Authors: Trittmann Jennifer K. , Almazroue Hanadi , Nelin Leif D.
Published: May 2022

Objective

Pulmonary hypertension (PH) predicts mortality in patients with bronchopulmonary dysplasia (BPD), and chronic hyperoxia exposure contributes to BPD‐PH disease progression. BPD‐PH is characterized by aberrant pulmonary vascular wall proliferation/remodeling, which occludes the microvascular lumen, increasing pulmonary vascular resistance. Endogenous nitric oxide (NO) production by pulmonary vascular endothelial cells promotes apoptosis of pulmonary vascular endothelial and smooth muscle cells. In vitro studies of human pulmonary microvascular endothelial cells (hPMVEC) have shown that dimethylarginine dimethylaminohydrolase‐1 (DDAH1) promotes NO‐mediated apoptosis. DDAH1 metabolizes the majority of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of endothelial NO synthase. Therefore, our hypothesis is that pharmacologically enhancing DDAH1 expression in hPMVEC will restore endogenous endothelial NO production.

Methods

hPMVEC were grown at 37 ̊C to ~70% confluence. Farnesoid X receptor (FXR) agonist, GW4064 (1.25 µM) was used to bind FXR element (FXRE) of the DDAH1 promoter to enhance transcription. hPMVEC were pre‐treated with FXR agonist for 24 hours and then exposed to 85%O2 or 21%O2 for an additional 24 hours. DDAH1 protein expression was analyzed by Western blot. Groups were compared by 1‐way ANOVA.

Results

In hPMVEC, hyperoxia exposure resulted in lower DDAH1 protein levels compared to hPMVEC grown in normoxia (N=6 in each group, p=0.003). Treatment with GW4064 prior to exposing to hyperoxia resulted in 4.3‐fold greater DDAH1 protein levels than in vehicle/85%O2 hPMVEC (N=6, p=0.044), and restored DDAH1 protein levels to 72% of that seen in the vehicle/21%O2exposed hPMVEC.

Conclusions

Chronic hyperoxia suppresses DDAH1 levels, and FXR agonist promotes DDAH1 recovery in hPMVEC. We speculate that promoting endogenous DDAH1 expression may prevent aberrant proliferation in the pulmonary vascular wall and thereby attenuate or prevent the vascular remodeling characteristic of pulmonary hypertension in BPD.
The Cardiorespiratory Coupling as a Background for Blood Pressure Decrease
Authors: Medviedieva Anastasiia , Shekh Vira , Iduh Mirabel D.
Published: May 2022

The role of cardiorespiratory coupling cannot be overemphasized. Cardiorespiratory coupling has many potential benefits in maintaining homeostasis. One of manifestation of cardiorespiratory coupling is respiratory sinus arrhythmia observed at respiratory rate 6 breaths/ minute. Multiple factors, including mechanical coupling, are responsible for the fluctuation of HR and BP. However, the fine mechanisms of the autonomic, cardiovascular, and respiratory interactions till now remains unclear.

A total of 156 international students were recruited from the V.N. Karazin Kharkiv National University. The sitting position was taken as a baseline according to requirements of the spirometry system. The ECG was continuously recorded at the rest and during slow breathing at 6 breaths/minute. The respiratory rate (RR) and tidal volume (TV) were recorded at the end of each stage for one minute using SPIROCOM pc based systems (XAI‐Medica, Ukraine). Systolic and diastolic blood pressure (SBP and DBP) were recorded by means automatic digital sphygmomanometer (OMRON EVOLV, Japan). The low and high frequency heart rate variability (LF and HF HRV) power were computed with CardioLab CS by fast Fourier transforming. The repeated measures MANOVA with Bonferroni correction (SPSS 22) was used to test for effects of slow breathing on HR, SBP, DBP, LnLF, LnHF, tidal volume (TV), minute ventilation (MV), and RR variables. Predictors of HR, SBP, DBP, TV, MV., and RR to slow breathing were determined using stepwise multiple linear regression analysis. Written informed consent was obtained from each participant.

In the current study the slowing respiratory rate to 6 breaths/min increased HR (P<0.001) (not shown), decreased SBP (P<0.001), decreased DBP (P=0.003), increased LnLF (P<0001), increased TV (P<0.001), and decreased RR (not shown, Р<0.001). The stepwise multiple linear regression analysis revealed that at rest HR and RR were negatively associated with LnHF power (Table 1, P<0.001 for both), while at slow breathing stage the HR was negatively associated with both LnHF and LnLF power (Table 2, P<0.001 and P=0.004, respectively). If the HF power is widely used as an index of cardiovagal activity, interpretation of the LF power is more controversal. We proceeded from our hypothesis that LF HRV power is a measure of baroreflex‐mediated sympathoinhibition. At rest both SBP and DBP were positively associated with LnLF by LnHF interaction, while at slow breathing stage they were negatively associated only with LnLF power. The resting TV and MV were positively associated with LnLF power, also, the MV with LnHF by height and BMI interaction. But at slow breathing stage only the height and BMI were independent variables remaining in these models (Table 2, Models 4 and 5).

Taken together, these findings indicate that the same autonomic variables control respiratory function in parallel with cardiovascular system.
An in‐Depth Analysis of the PAK1 Autophagy Signaling Pathway in H9C2 Cardiomyoblasts
Authors: Geffken Shawn , Guha Anoushka , Jaiswal Pooja , Kobayashi Tamayo , Kobayashi Satoru , Liang Qiangrong
Published: May 2022

P21‐activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1‐dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5‐12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.
Mutation in the 5'SL Region of Collagen I mRNA Attenuates Diastolic Dysfunction in Male, but not Female, Mice Following 28 Weeks of Feeding a High Fat High Sucrose Diet
Authors: Russell Jacob , Bender Shawn B. , Ramirez‐Perez Francisco , Woodford Makenzie , Ruff Margot , Brenner David , Yoshida Tadashi , DeMarco Vincent , Martinez‐Lemus Luis , Padilla Jaume , Chandrasekar Bysani
Published: May 2022

Introduction

The La ribonucleoprotein 6 (LARP6), an RNA binding protein, increases type I collagen expression by regulating mRNA stability and translation. This is mediated via its interaction with the 5’ stem loop (5’SL) structure in collagen I mRNA. Disrupting this interaction by mutating the 5’SL region has been shown to reduce fibrotic remodeling in liver. Therefore, we tested the hypothesis that this interaction plays a role in cardiac fibrosis and stiffening in diet induced obesity in 5’SL mutant and wild‐type littermate mice. In fact, obesity and hypertension promote remodeling and cardiac stiffening due to excess collagen deposition, fibrosis, and microvascular dysfunction, that ultimately lead to diastolic dysfunction.

Methods

Male and female 5’SL mice and wild‐type (WT) littermates were fed a high fat high sucrose (HFHS) or control diet starting at 6 weeks of age. After 28 weeks of feeding, mice were assessed for cardiac function by echocardiography. Hearts were collected and a transverse section of the left and right ventricle was fixed for histology. RNA was isolated from the left ventricle and submitted for mRNA sequencing and qPCR.

Results

Following 28 weeks of a HFHS diet, male and female mice showed increased body weight, cardiac hypertrophy (heart weight normalized to tibia length), and diastolic dysfunction. However, the 5’SL mutation attenuated the impaired diastolic function in male, but not female mice. Surprisingly, cardiac fibrosis was not altered by the HFHS diet. RNA sequencing revealed that HFHS diet altered gene expression associated with glucose and ketone metabolism, as well as inflammation, in male WT littermates. However, the 5’SL mutation enriched gene networks associated with vascular development and angiogenesis.

Conclusions

Chronic HFHS feeding leads to increased body weight, hypertrophic cardiomyopathy, and diastolic dysfunction in both male and female mice. However, the 5’SL mutation blunts dysfunction only in male mice, demonstrating sex‐dependent cardioprotective effects.
DGKζ interacts with ERK3 and counteracts the promoting role of ERK3 in lung cancer migration
Authors: Myers Amanda , Popp Katherine , Long Weiwen
Published: May 2022

Extracellular‐Regulated Kinase 3 (ERK3) promotes cell migration and invasion in many cancers, including lung cancer. However, the mechanisms by which this atypical mitogen‐activated protein kinase (MAPK) is regulated remain poorly understood. In a yeast two‐hybrid assay, ERK3 was shown to interact with diacylglycerol kinase ζ (DGKζ), an enzyme which converts diacylglycerol to phosphatidic acid. This interaction has been confirmed by co‐immunoprecipitation and occurs through the C34 domain of ERK3 and the N‐terminus and C1 domain of DGKζ. By immunofluorescence, these proteins were shown to mainly co‐localize at protruding regions of the plasma membrane in cells. Interestingly, DGKζ reduces cell migration in lung cancer cell lines in a kinase‐independent manner. While ERK3 promotes cell motility in lung cancer cell lines, DGKζ overexpression negates this effect entirely in the transwell assay. Further, DGKζ reduces ERK3 accumulation at protrusions of the cell membrane but cannot alter localization of a mutant ERK3 lacking the C34 domain. In conclusion, we have identified DGKζ as a new interaction partner and inhibitor of ERK3 in regulating lung cancer cell migration.
Screening of Machine Learning Predicted Inhibitors of Mur‐E Ligase
Authors: Smith Behrgen , Debruine Anna , Young Keoni , Wegner Everen , Giese Ryan , Connelly Evan , Haensgen Michaela , Klatt Emma , Callahan Aidan , Schmidt Keagan
Published: May 2022

Antibiotic resistance is an ever‐growing threat, which necessitates the development of novel antibiotics. Peptidoglycan (PTG) is an ideal target for antibiotic development as it is necessary for the structural integrity of the cell and is unique to bacterial cells. Mur‐E, a cytoplasmic ligase, is an integral component of PTG synthesis and shares many conserved structures with PTG ligases in other bacterial species. Thus, inhibitors of Mur‐E are likely to be effective broad‐spectrum antibiotics. Mur‐E's role in PTG synthesis is to add meso‐A2pm or L‐lysine to a nucleotide precursor of PTG, UMAG; this reaction is facilitated by ATP hydrolysis. Residues N449, R451, K157, E220, D392 were identified in a mutagenesis study as having significant impacts on enzyme kinetics. This project combines the knowledge of each residue’s known impact on kinetics with an analytical, dynamics‐based investigation of protein structure using Essential Sit Scanning Analysis (ESSA) and ClustENMD. These two methodologies implement elastic network models of proteins to predict allosteric pockets and alternate conformations of the protein, respectively. This will give insight into which pockets are likely to be effective drug targets based on their impact on enzyme kinetics and protein dynamics; these pockets will be evaluated using a library of predicted ligands for Mur‐E to identify potential inhibitors with AutoDock Vina. By evaluating ligand binding sites and affinities, probable antibiotics can be determined. Targeting residue pockets that facilitate key Mur‐E functions, like ATP binding and substrate addition, will produce the most effective antibiotic treatment by compromising PTG production.
Inhibition of Peroxiredoxin 6 PLA2 Activity Decreases Oxidative Stress and the Severity of Acute Lung Injury in the Mouse Cecal Ligation and Puncture Model
Authors: Chatterjee Shampa , Dodia Chandra , Tao JianQin , Feinstein Sheldon I. , Fisher Aron B.
Published: May 2022

Introduction

ALI is associated with bacterial infection (sepsis), either as an initiating factor or as a secondary complication. Activation of polymorphonuclear leucocytes (PMN) recruited into the lungs causes the release of free radicals and reactive oxygen species (ROS) that are responsible for many of the clinical manifestations of ALI. Since NADPH oxidase (NOX2) is the major source of ROS (i.e., superoxide anion) in PMN, NOX2 blockade has been proposed for the treatment of Acute Lung Injury (ALI). We reasoned that NOX2 blockers would be counter‐productive in ALI associated with bacterial infection by interfering with the bactericidal activity of PMN.

Hypothesis

Phospholipase A2 inhibitory peptide (PIP‐2) that inhibits NOX2 activation (by preventing activation of Rac, a necessary NOX2 co‐factor) would inhibit the bactericidal activity of PMN and thereby limit clearance of bacteria from the lung; we posited that this effect of PIP‐2 could be overcome by the administration of appropriate antibiotics.

Methods

Using the cecal ligation and puncture (CLP) model of ALI associated with sepsis, we investigated the effect of inhibiting NOX2derived ROS production by administration of PIP‐2.

Results

The number of colony‐forming bacteria in lungs and peritoneal fluid of mice with CLP approximately doubled at 18 h after intravenous treatment with 2 µg PIP‐2 /gram body weight (wt). Treatment with 10 µg PIP‐2/g body wt resulted in 100% mortality within 18 hr. Antibiotic treatment abolished both the increase in lung bacteria with low dose PIP‐2 and the increased mortality with high dose PIP‐2. Treatment with PIP‐2 in the presence of antibiotics resulted in significantly improved lung histology, decreased PMN infiltration, decreased lung fluid accumulation, and decreased oxidative lung injury compared to antibiotics alone.

Conclusion

Administration of PIP‐2 provides partial protection against lung injury in a model of ALI due to bacterial infection while concurrent antibiotic treatment abolishes the deleterious effects of PIP‐2 on lung bacterial clearance. These results suggest that addition of PIP‐2 to the antibiotic regimen would be beneficial for treatment of ALI associated with bacterial infection.
Altering the Stability of an Upstream Domain Affects the Proteasome’s Ability to Unfold a Downstream Domain
Authors: Lago Athena V. , Kraut Daniel A.
Published: May 2022

The Ubiquitin‐Proteasome System (UPS) is responsible for the bulk of protein degradation in eukaryotes. The 26S proteasome is the ATP‐dependent protease component of the UPS that degrades misfolded, nonfunctional, and regulatory proteins. Substrates are targeted to the proteasome via polyubiquitination, followed by engagement, translocation, unfolding, and degradation. We are specifically interested in partial or incomplete degradation of substrates by the proteasome. Partial degradation can occur when the proteasome stalls on a difficult‐to‐unfold protein domain and then releases a portion of the protein as a stable fragment. Several “slippery substrates” have been identified, but the mechanisms that determine whether a substrate is fully or partially degraded remain unclear. We study the unfolding ability of the proteasome with a construct that contains an N‐terminal degron, an easy‐to‐unfold barnase domain, and a harder‐to‐unfold DHFR domain. By measuring the extent to which DHFR is either fully degraded or released as a fragment, we can determine the proteasome’s unfolding ability. Surprisingly, we have found that destabilizing the barnase domain via an L89G mutation (which has been reported to destabilize barnase by 7 kcal/mol) increases the ability of the proteasome to unfold the DHFR domain, even though no mutation or stability changes were made to DHFR. This result suggests that as the proteasome moves sequentially through a substrate, the stability of domains that it first encounters may affect the fate of downstream domains.
Bile acids directly bind and allosterically regulate the epithelial sodium channel
Authors: Wang Xueping , Tomilin Victor , Ertem Merve , McKernan Abigail , Lei Xiaoguang , Pochynyuk Oleh , Kashlan Ossama B.
Published: May 2022

Bile acids are critical for the digestion and absorption of dietary fats and have important roles as signaling molecules. Bile acids are normally restricted to the enterohepatic circulation, but can be elevated in the blood and urine of patients with advanced liver diseases. The epithelial sodium channel (ENaC) mediates Na+ transport in the biliary epithelium, where it regulates bile viscosity, and in the aldosterone‐sensitive distal nephron, where it regulates extracellular fluid volume. We previously showed that bile acid regulation of ENaC depends on the position and stereochemistry of specific functional groups, but not on their chemico‐physical properties. To determine whether bile acids regulate ENaC in ex vivo native tissues, we performed patch clamp experiments on isolated split open renal tubules from mice, using recording pipettes backfilled with taurocholic acid (t‐CA) or taurohyodeoxycholic acid (t‐HDCA). Consistent with in vitro experiments, t‐CA increased ENaC open probability (PO) by 2‐fold while t‐HDCA decreased ENaC PO by nearly two‐thirds. To determine whether bile acids regulate ENaC through direct binding, we performed photoaffinity labeling experiments. We employed a bile acid derivative (p‐DCA) with diazirine and terminal alkyne groups that facilitate uv‐crosslinking and affinity purification, respectively. Using cultured Fisher Rat Thyroid cells transfected with ENaC, we found that the deoxycholic acid derivative (p‐DCA) crosslinked to the α, β, and γ subunits. To determine whether crosslinking was specific, we performed experiments in the presence of competing bile acids. Crosslinking to the β subunit was significantly reduced in the presence of deoxycholic acid (DCA). Crosslinking to the γ subunit was significantly reduced by taurocholic acid (t‐CA). These data suggest direct binding and multiple sites. To determine the effect of subunit composition on regulation, we measured the effect of t‐CA on Li+ currents in Xenopusoocytes expressing different functional ENaC subunit combinations. Channels composed of α subunits alone were strongly activated by t‐CA, followed by αβγ‐ and αβ‐ channels, suggesting that the α subunit is sufficient for ENaC sensitivity to t‐CA. Our data are consistent with bile acids directly binding all three ENaC subunits at multiple sites. Bile acid moieties provide for selective preferences for each of the sites, resulting in distinct effects for each bile acid.
The impact of walking posture on trabecular structure in the femur of Rattus norvegicus
Authors: Campillo America M. , Kuo Sharon , Foster Adam D. , Ryan Timothy M.
Published: May 2022

Bone responds to mechanical loads through growth. This response to external loading has been important for understanding skeletal form and function relationships. However, uncertainty still exists regarding how different locomotor behaviors, (e.g., bipedalism, quadrupedalism) are reflected in bone morphology. This study assesses the effects of locomotor posture on femoral bone structure in a rodent model (Rattus norvegicus). The study individuals come from a previously‐executed experiment (Foster, 2019) that utilized a treadmill‐mounted harness to induce bipedal walking postures in rats over 12‐weeks. The sample includes individuals from two experimental groups plus a control: bipedal walking, quadrupedal walking, and no exercise controls. Proximal and distal femora were microCT scanned at 20µm and trabecular bone volume fraction (BV/TV) and degree of anisotropy (DA) were analyzed and compared among groups to assess the impacts of differences in hip loading. In the proximal femur, the bipedal group had higher DA inferior to the neck and greater trochanter than the quadrupedal group, suggesting more unidirectional loading in bipeds. However, the quadrupedal group had greater BV/TV throughout the proximal metaphysis; particularly at the greater trochanter, which may be due to a higher demand for hip extension. In the distal femur, the bipedal group had higher mean BV/TV (0.39) when compared to the quadrupedal group (0.36), with a larger concentration of BV/TV in the medial condyle. These results suggest that the femoral epiphyses are responsive to changes in hip postural angle due to locomotion, however cortical bone structure and a larger sample size may be more informative.
Elucidating the Role of N‐cadherin during Trigeminal Gangliogenesis
Authors: Halmi Caroline , Taneyhill Lisa
Published: May 2022

Cadherin‐based adhesion is critical to the formation of multicellular tissues, including the cranial sensory ganglia of the peripheral nervous system. Initial assembly of the cranial trigeminal ganglion, which houses the cell bodies for the trigeminal nerve, relies on the coalescence of N‐cadherin (N‐cad)‐expressing placode cell‐derived neurons and Cadherin‐7‐expressing neural crest cells. The trigeminal ganglion has a role that is primarily sensory, transmitting information on pain, touch, and temperature from different facial regions back to the central nervous system. Proper survival and differentiation of trigeminal neurons relies, in part, upon binding of neurotrophic and other growth factors secreted from target tissues to their cognate receptors found on peripheral axons. These complexes are then trafficked to neuronal cell bodies, resulting in the activation of downstream signaling pathways. Prior studies have shown that N‐cad internalization and trafficking are required for proper neuronal outgrowth. Moreover, N‐cad knockdown in chick trigeminal placode cells leads to early defects in trigeminal ganglion assembly by increasing placodal neuron dispersal. Later functions for N‐cad in chick trigeminal gangliogenesis, however, remain obscure. We hypothesize that N‐cad controls trigeminal ganglion development through the regulation of multiple signaling pathways. To address this, we performed morpholino‐mediated knockdown of N‐cad in chick trigeminal placode cells followed by immunohistochemistry and biochemistry to evaluate trigeminal gangliogenesis. Our preliminary data show that N‐cad depletion has a sustained negative impact on the trigeminal ganglion through cell and non‐cell autonomous effects, resulting in decreased ganglion size, aberrant nerve outgrowth and branching, and reduced levels of Cadherin‐7, neurotrophins and their receptors, and Smad family members. Future studies will aim to elucidate the mechanisms by which N‐cad controls trigeminal ganglion development via these molecules and pathways, including experiments designed to examine protein trafficking in trigeminal neurons. Taken together, our findings reveal potential adhesion‐dependent and ‐independent functions for N‐cad in the trigeminal ganglion, which will aid in the understanding of its tissue‐specific roles. This knowledge will shed light on the etiology of disorders characterized by abnormal neural crest cell and placode cell development and those affecting the peripheral nervous system.
Brainstem BBSome Regulates Glucose Metabolism and High Fat Diet Sensitivity
Authors: Laule Connor , Guo Deng , Rahmouni Kamal
Published: May 2022

Bardet‐Biedl syndrome (BBS) proteins form a protein trafficking complex (BBSome) that regulates the composition of membrane proteins and contributes to neuron function. Patients harboring mutations in any BBS gene results in BBSome dysfunction and the development of metabolic complications including obesity and type 2 diabetes. Studies from our lab and others indicate that hypothalamic BBSome is a critical regulator of metabolic homeostasis, however the role of the BBSome in the brainstem is unknown. To determine whether hindbrain BBSome contributes to metabolic regulation, we crossed Phox2bCre mice with BBS1fl/fl mice (Phox2bCre/BBS1fl/fl) yielding BBS1 deletion specifically in brainstem nuclei. BBS1 deletion exclusively in hindbrain nuclei was validated by breeding these mice on a tdTomato Cre reporter background. Male Phox2bCre/BBS1fl/fl mice maintained on standard chow diet did not exhibit energy imbalance as evident by comparable body weight (37.2±2.8g vs. 35.8±4.4g), body composition (fat 20.8±2.8% vs 15.6±4.1%, lean 60.6±2.5% vs 64.2±3.0%), and food intake (3.5±0.4g/day vs 3.8±0.5g/day) relative to litter‐mate controls at 20 weeks of age. Interestingly, hindbrain BBSome disruption impairs glucose clearance and induces insulin resistance suggested by glucose (AUC 36,323±3027 vs 26,129±1972) and insulin (AUC 11,749±1678 vs 7,737±1559) tolerance tests. Strikingly, Phox2bCre/BBS1fl/fl male mice exhibit resistance to diet‐induced obesity indicated by decreased body weight (45.0±1.8g vs. 54.1±1.3g) when maintained on a high fat/high sugar diet independent of body composition (fat 42.5±1.6% vs 40.1±2.1%, lean 45.5±0.7 vs 48.4±1.5%) and food consumption (2.7±0.2g/day vs 3.0±0.1g/day). Both Phox2bCre/BBS1fl/fl and littermate controls developed diabetes when exposed to an obesogenic diet as indicated by glucose (AUC 63,645±4438 vs 62,477±2654) and insulin (AUC 14,303±3446 vs 16,406±2160) tolerance tests. These findings suggest that brainstem BBSome critically regulates glucose metabolism, but not energy balance, in mice maintained on a standard diet regimen. Furthermore, our studies indicate that hindbrain BBSome contributes to the development of diet‐induced obesity without effecting glucose handling and food intake.
Impact of Branched Chain Amino Acid Supplementation on Muscle Lipid Accumulation and Mitochondrial Metabolism
Authors: Ryan Caitlin E. , Patel Kruti , Muralidaran Vaishna , Surugihalli Chaitra , Bhattacharjee Parama , Sunny Nishanth E.
Published: May 2022

Cross talk between branched‐chain amino acids (BCAAs; valine, leucine and isoleucine), mitochondrial metabolism and lipid accumulation have been shown to occur in several tissues, including skeletal muscle. In fact, defects in BCAA metabolism and their degradation networks coexist with defects in lipid metabolism in insulin resistant muscle. Here, we hypothesized that the relationship between BCAAs and lipid metabolism is due to the ability of BCAAs to modulate mitochondrial oxidative metabolism and lipid accumulation in the muscle. Mice (C57BL/6N) were randomly assigned to a low‐fat diet (LFD; 10% fat kcal), LFD supplemented with BCAAs (LFBA; 150% BCAA), high‐fat diet (HFD; 60% fat kcal) or HFD supplemented with BCAAs (HFBA; 150% BCAA) for 12 or 26 weeks (n = 8‐11 per group). Following the dietary treatments, leg muscle tissue were collected and flash frozen in liquid nitrogen, under feeding and overnight fasting conditions. Lipid accumulation was evaluated by analyzing muscle triglycerides, Oil‐Red‐O staining, and gene expression profiles. Mitochondrial metabolism and BCAA degradation were evaluated using a combination of gas‐chromatography/mass‐spectroscopy based targeted metabolomics and western blot analysis. Levels of BCAAs in the muscle showed significant correlations to their corresponding keto‐acids (p ≤ 0.01). However, supplementation of BCAAs did not affect the levels of mitochondrial tricarboxylic acid (TCA) cycle intermediates or amino acids in the muscle. Further, mitochondrial proteins involved in oxidative phosphorylation also remained similar between non‐supplemented and BCAA supplemented muscle. Muscle tissue from HFD mice had higher lipid accumulation (mg/g ± SEM) (LFD; 8.22 ± 0.91, HFD; 11.03 ± 1.08; p ≤ 0.05), compared to their LFD counterparts. Interestingly, BCAA supplementation for 12‐weeks along with the HFD blunted muscle lipid accumulation (LFBA; 8.62 ± 1.12, HFBA; 9.76 ± 0.75), which was also evidenced from Oil Red‐O staining of the muscle (p = 0.07). A similar lowering of lipid accumulation (p ≤ 0.05) was evident between the HFD vs. HFBA groups, following 26‐weeks on the diets, as quantified by Oil Red‐O staining. Further, the expression of genes involved in lipid accumulation/synthesis Srebp1c (p ≤ 0.05) and Acc1 (p ≤ 0.1) were lower in the 12‐week LFBA vs. their LF counterparts, under fed conditions. Taken together, our results show that BCAAs have the ability to blunt muscle lipid accumulation without altering mitochondrial oxidative metabolism in the muscle.
Development of New Assays for the Simultaneous Measurement of DNA Double‐Strand Break Repair by Multiple Pathways
Authors: Valdez‐Oranday Diego F. , Lewis L. K.
Published: May 2022

Cellular DNA is damaged by both endogenous and exogenous sources. Among the different types of DNA lesions that are formed, double‐strand breaks (DSBs) are considered to be the most consequential lesions. They can cause genomic instability and cellular death if not repaired via one of two pathways: nonhomologous end‐joining (NHEJ) or homologous recombination (HR). Deficiency of DSB repair is implicated in several congenital diseases and cancer‐prone disorders. In the budding yeast Saccharomyces cerevisiae NHEJ is accomplished by the actions of the Yku, Mrx, and Dnl4 protein complexes. Recent experiments in this lab have demonstrated that, in contrast to Yku and Dnl4, the Mrx complex is required for NHEJ repair of some DSB end structures but not others. The major goal of the current project is to characterize the role of Mrx and other protein complexes in repair by NHEJ and HR using newly developed plasmid and PCR fragment‐based assays. Experiments performed for the project demonstrated that repair of DSBs with 3′ single‐stranded DNA overhangs was reduced in yku70, mre11 and dnl4 mutants; however, the defect in NHEJ was strongly suppressed when EXO1, encoding a major 5′‐to‐3′ exonuclease, was inactivated in mre11 cells, i.e., in mre11 exo1 double mutants. By contrast, absence of the Sgs1‐Dna2 nuclease‐helicase complex (in mre11 sgs1 mutants) had no effect. NHEJ repair of DSBs with 5′ single‐stranded DNA overhangs was not changed by inactivation of EXO1 or SGS1. These findings implicate Mrx as a potential protector of DSB ends with 3′ overhangs during NHEJ repair and Exo1 as the main degradative nuclease. Additionally, we have developed new plasmid‐ and PCR fragment‐based repair assays that can analyze NHEJ and HR simultaneously using auxotrophic markers and type IIS restriction endonucleases. We anticipate that the new methods for simultaneous measurement will permit exploration of pathway choice in ways not previously possible.
Specific Ablation of Jak2 from CD11c+ Cells Attenuates Salt‐Sensitive Hypertension through an ENaC‐Dependent Mechanism
Authors: Saleem Mohammad , Aden Luul , Pitzer Ashley , Elijovich Fernando , Laffer Cheryl , Pakala Suman , Ishimwe Jeanne , Kleyman Thomas , Kirabo Annet
Published: May 2022

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase‐dependent formation of immunogenic isolevuglandin (IsoLG)‐protein adducts leading to inflammation and salt‐sensitive hypertension. We hypothesized that expression of Jak2 specifically in antigen presenting myeloid cells contributes to salt‐sensitive hypertension in an ENaC dependent mechanism. Profiling whole transcriptome using RNA‐Seq and analyzing the gene differential expression patterns in human monocytes reveals that high salt treatment upregulates genes of the Jak/STAT pathway, and the downstream regulators suppressor of cytokine signaling (SOCS) genes. Male and female mice lacking Jak2 in CD11c+ cells developed blunted hypertension (123.8±4.7) during the high salt feeding phase of the N‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME)/high salt model of salt‐sensitive hypertension compared to the wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention in response to high salt‐feeding when compared to the wildtype littermate controls. We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ, and serum/glucocorticoid regulated kinase 1 (SGK1) in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less renal infiltration of effector memory T cells (TEM), less aortic infiltration of CD11c+ cells with less expression of CD86, and less production of IsoLGs and IL1‐beta. These results indicate that dendritic cell Jak2 plays an important role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.
Overexpression of the Neurospora crassa Transcription Factor fsd‐1 Inhibits Mating
Authors: Smith Hannah , Gera Kaitlyn , Hutchison Elizabeth
Published: May 2022

The filamentous fungus Neurospora crassa can undergo both asexual and sexual reproduction. However, little is known about the molecular mechanisms that regulate the N. crassa female sexual development cycle. Previous research has shown that deletion strains of the transcription factor fsd‐1 have delayed development of female reproductive structures and are sterile. fsd‐1 is transcribed into three different transcripts, which differ by the length and intron/exon structure of their 5’ untranslated region, and these transcripts vary in expression level during the N. crassa life cycle. fsd‐1 controls the development of female reproductive structures in N. crassa, which affects many downstream processes. The goal of this project is to phenotypically characterize the reproductive ability of strains overexpressing fsd‐1, for each of the three transcripts, and we hypothesize that fsd‐1 overexpression will affect the reproductive ability of these strains. Characterization of the changes in transcriptional expression caused by fsd‐1 overexpression will give us insight into the fsd‐1 regulon. Using Illumina RNA sequencing, we mapped the genome‐wide transcriptional changes that occur as a result of fsd‐1 overexpression. Analysis of the reproductive abilities of strains that overexpress fsd‐1 showed that these strains can produce spores, but that the spores do not germinate. Our RNAseq and phenotype experiments revealed that fsd‐1 regulates processes including melanin synthesis, cell wall formation, and ascospore viability, and plays a key role in sexual development.
Infantile Neurodegeneration results from Mutants of 17β‐Hydroxysteroid Dehydrogenase Type 10 rather than Aβ‐Binding Alcohol Dehydrogenase
Authors: yang song‐yu , He Xue‐Ying , Dobkin Carl , Brown W. Ted
Published: May 2022

Type 10 17β‐hydroxysteroid dehydrogenase (17β‐HSD10), a mitochondrial homo‐tetrameric multifunctional protein encoded by the HSD17B10 gene is necessary for brain cognitive function. Missense mutations on this gene resulted in HSD10 mitochondrial disease, an inborn error in isoleucine metabolism. A 5‐methylcytosine hotspot in this gene underlying a 388C ̵T transition leads to the HSD10(p.R130C) mutant responsible for about half cases of infantile neurodegeneration. Less females suffers with this disease and usually has milder clinical manifestation due to the X‐inactivation. The binding capability of this dehydrogenase to Aβ‐peptide may play a role in senile neurodegeneration, namely Alzheimer disease, but it appears unrelated to infantile neurodegeneration. Unfortunately, this research field has been interfered by reports of the Aβ‐peptide binding alcohol dehydrogenase (ABAD) or endoplasmic reticulum‐associated Aβ binding protein (ERAB). Both reflect some dubious features of 17β‐HSD10. It is clarified here that ERAB is merely a subunit of 17β‐HSD10, having a fictitious association with the endoplasmic reticulum. 17β‐HSD10 exhibits L‐3‐hydroxyacyl‐CoA dehydrogenase activity so also known as short chain 3‐hydorxyacyl‐CoA dehydrogenase (SCHAD), but it is not involved in the ketone body metabolism as reported for ABAD’s function in the cellular response to nutritional stress. Furthermore, the reported generalized alcohol dehydrogenase data underlying the ABAD activity could not be based upon valid reproducible experiments, and the rediscovery of its mitochondrial localization was preceded by unreferenced 17β‐HSD10 research. Clarification of unreliable ABAD/ERAB data may invigorate this research field, and open new approaches to the understanding and treatment of the HSD17B10gene related disorders including infantile neurodegeneration and Alzheimer’s disease.
Hypoglossal Motoneuron (XII MN) Survival Over the Lifespan in Pompe Disease
Authors: Barral Brian E. , Singer Michele L. , Rana Sabhya , Lavassani Emmanuelle , Pope Megan K. , Byrne Barry J. , Fuller David D.
Published: May 2022

Glycogen Storage Disease II, more commonly known as Pompe disease, is a lysosomal storage disease characterized by a partial or complete loss of acid α‐glucosidase (GAA) activity. Lingual impairment and sleep disordered breathing are common symptoms of Pompe disease with a potential role of XII MN dysfunction in both cases. Through innervation of the tongue muscles, XII MNs contribute to swallowing, speech, and maintenance of upper airway patency. Studies of Pompe (Gaa‐/‐) mice have found molecular and histological markers of neurodegeneration, and a recent case study of a patient with late‐onset Pompe disease revealed atrophied and possible degenerating XII MNs. However, whether the absence of GAA activity in Pompe disease directly leads to death of XII MNs is not definitively established. Because the preponderance of evidence indicates progressive neurodegeneration in Pompe disease, here we tested the hypothesis that when XII MNs do not express GAA activity, there will be a progressive loss of these cells over the lifespan. A novel transgenic mouse model (ChAT‐cre+/+●Gaa+/‐●eYFP+/+●Flox‐Gaa+/+) was generated in which GAA activity is absent in ChAT positive neurons, which also carry a yellow fluorescent protein (YFP) marker. Thus MNs in this mouse model do not express GAA, but skeletal myofibers have normal GAA expression. Brainstem tissue was harvested in young (1‐3 months of age) and advanced age mice (21‐24 months). Tissues were cryosectioned at 20 μm increments across the entire XII motor nucleus. Sections were slide mounted and visualized under fluorescence microscopy to detect YFP. The XII motor nucleus was identified based on anatomical landmarks and MNs were identified based on location, size, morphology, and YFP fluorescence. Preliminary MN counts across the rostral‐caudal extent of the XII nucleus (n=3 per group) indicate an approximately 7% decline in detectable YFP‐positive XII MNs over the age span (young: 658±19; old 617±28). Further, the histological appearance of XII MNs showed striking differences between the two age groups. The MNs from older mice showed the prototypical hallmarks of neuronal lysosomal storage disease including a markedly enlarged soma and extensive vacuolization. These preliminary findings are consistent with the hypothesis that absence of GAA in XII MNs leads to a histopathological appearance and neurodegeneration. Ongoing studies are evaluating XII MNs in age matched wild type mice.