bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of Multiple E3 Ubiquitin Ligases in Gastrocnemius Muscles from Mice
Authors: Hughes David C. , Baehr Leslie M. , Waddell David S. , Bodine Sue C.
Published: May 2022

E3 ubiquitin ligases are commonly studied in skeletal muscle under atrophy‐inducing conditions. MuRF1 is a recognized marker of muscle atrophy and we have recently identified Fbxl22 to be involved in the process of denervation‐induced muscle atrophy. Thus, we sought to investigate the role of MuRF1 and Fbxl22 together in the process of skeletal muscle atrophy. Male C57BL/6 mice (16 to 20‐week old) were subjected to in vivo electroporation with either a mouse specific MuRF1 or Fbxl22 plasmid (20ug) in the gastrocnemius (GA) muscles with the contralateral leg serving as a vector control (EV). An additional cohort of mice was electroporated with MuRF1 and Fbxl22 plasmids simultaneously. After 7 days, lateral (LGA) and medial (MGA) gastrocnemius muscles were collected, weighed and sectioned for cross‐sectional area (CSA) and GFP visualization. Western blotting was performed on sarcoplasmic and myofibrillar fractions to determine levels of ubiquitin and sarcomeric proteins in the transfected GA muscles.

MuRF1 overexpression (OE) resulted in significant loss of total GA muscle mass (‐8.8%) after 7 days alone. In comparison, Fbxl22 OE led to no loss in total GA muscle mass, although significant reductions in LGA mass (‐3.4%) were observed. Combined OE of MuRF1 and Fbxl22 together resulted in significant loss of GA muscle mass (‐5.5%) but the level of atrophy was not as great as compared to MuRF1 OE alone. In transfected LGA muscles, total ubiquitin levels were significantly elevated in the Fbxl22 OE and combined E3 ligase group for the sarcoplasmic fraction. In the myofibrillar fraction of LGA transfected muscles, only the combined E3 ligase group displayed increased total ubiquitin levels after 7 days. In transfected Fbxl22 LGA muscles, we observed significant increases in vimentin, alpha‐actinin and Troponin T in the sarcoplasmic fractions. Gene manipulation of the E3 ubiquitin ligases, MuRF1 and Fbxl22 in basal skeletal muscle resulted in skeletal muscle atrophy and divergent changes in ubiquitination and sarcomeric proteins were observed following E3 overexpression. Future studies will seek to identify potential unique and shared substrates targeted by Fbxl22 and MuRF1 for ubiquitination.
DAP5 Binding to the FGF‐9 mRNA 5’ UTR is Structurally‐Dependent
Authors: Whittaker Amanda M. , Goss Dixie J.
Published: May 2022

Eukaryotic mRNA translation is typically modulated by eukaryotic initiation factor 4E (eIF4E) binding to the 5’‐m7G cap. However, under the hypoxic conditions within the cancer cell, eIF4E is segregated by 4E binding protein. Thus, cap‐independent recruitment of the 40S ribosome to the 5’ untranslated region (5’ UTR) of the mRNA is required for tumorigenesis. Fibroblast growth factor 9 (FGF‐9) is one such angiogenic growth factor upregulated in a cap‐independent manner in breast and colorectal cancer cells. Previously, our lab has demonstrated that binding of death‐associated factor 5 (DAP5), a eukaryotic initiation factor, to the FGF‐9 5’ UTR directly impacts cap‐independent translational efficiency. However, the exact binding site of DAP5 within the 5’ UTR is unknown. RNA secondary and tertiary structure within non‐coding regions has been demonstrated to play a role in cap‐independent translational efficiency; therefore, we hypothesized that specific structures act as eukaryotic initiation factor binding sites. Using SHAPE‐sequencing data, we reveal that the 5’ UTR of FGF‐9 is highly structured and possesses a single‐stranded region near the start codon. Through SHAPE footprinting and toeprinting experiments, we also demonstrate that DAP5 binds along a single face of the FGF‐9 5’ UTR and contacts the 3’ single‐stranded region. Our lab had previously reported that cap‐independent translation of FGF‐9 does not require a free 5’ end, suggesting little to no ribosomal scanning for the start codon occurs. Our current data demonstrate that initiation factor binding is structurally‐dependent and suggest that limited ribosomal scanning is due to DAP5‐dependent restructuring of the 5’ UTR to bring the start codon within striking distance of the 40S ribosome. We are currently examining whether cap‐independent upregulation of other tumorigenic transcripts follows a similar pattern.
5‐Fluorouracil and Gemcitabine Alter Nuclear Transport
Authors: LaMark Emily , Nickle Audrey , Resendes Karen
Published: May 2022

The GTPase, Ran, is a major regulator of nuclear transport and disruption of its established gradient disrupts nuclear export and leads to cell death through the accumulation of pro‐apoptotic factors. The chemotherapy 5‐Fluorouracil (5‐FU) increases nuclear pore permeability causing Ran to leak into the cytoplasm, disrupting the gradient and in turn nuclear export, leading to cell death of HeLa cervical cancer cells. Our previous work demonstrated that combining 5‐FU with topoisomerase inhibitors can have a greater total impact on disruption of the Ran gradient and accumulates proteins in the nucleus. Gemcitabine (GEM) is widely used when treating pancreatic cancer; yet a resistance is common. However, combination of GEM with nuclear export inhibitors can help overcome this problem. Therefore, we sought to determine if combination of 5‐FU and GEM could impact nuclear export to help overcome GEM resistance. Our initial studies found that 5‐FU+GEM further decreased nuclear Ran in HeLa cells and lead to increased cell death in combination over GEM alone. Therefore, we transitioned to exploring the effects of 5‐FU+GEM on disrupting the Ran gradient in PANC1 pancreatic cells. Both 5‐FU and GEM individually and in combination decreased the nuclear Ran levels by 15%, 17%, and 23% respectively. This disruption of nuclear export may help overcome GEM resistance in PANC1 cells. Specifically, our ongoing work is exploring the impact of 5FU+GEM on the nuclear retention of the tumor suppressors p27 and p21 as well as cell viability to identify a possible mechanism for the impact of the 5‐FU+GEM combination on apoptosis. Our work helps support a possible mechanism for overcoming GEM resistance in Panc1 cells and identifies a potential combination treatment with 5‐FU for use against pancreatic cancer.
nTS Glutamate Signaling in the Chemoreflex Axis of an Alzheimer's Disease Rat Model
Authors: Tipton Robert K. , Humphrey Chuma M. , Scales Dorothy M. , Ostrowski Daniela , Ostrowski Tim D.
Published: May 2022

Respiratory dysfunction is commonly observed in Alzheimer’s disease (AD) and likely due to neuronal alterations of the respiratory network in the brain. The streptozotocin (STZ) ‐induced AD model mimics respiratory problems seen in humans and exhibits neuronal hyperactivity in the nucleus tractus solitarii (nTS), the central integration site for respiratory afferents in the brainstem. This ongoing study looks at the changes of glutamate signaling within the chemoreflex axis as a possible mechanism for nTS hyperactivity and respiratory dysfunction in STZ‐AD.

AD was induced by intracerebroventricular injection of 2 mg/kg STZ in 6‐week old male Sprague Dawley rats. After 14 days, caudal nTS neurons within close proximity of DiI‐labeled chemoafferent terminals were recorded using the patch clamp technique. Electrical stimulation of the tractus solitarii (TS) generated glutamatergic excitatory postsynaptic currents (EPSCs) that were compared between experimental groups. Consistent with enhanced glutamatergic signaling, TS‐EPSC amplitude was increased in STZ‐AD. High TS stimulation frequency induced a time and frequency dependent depression that was similar between groups, indicating no change in presynaptic vesicle turnover or postsynaptic receptor desensitization. On the other hand, asynchronous EPSCs following high frequency stimulation seemed enhanced in STZ‐AD, supporting an increased excitatory tone in the nTS. Immunohistochemical analysis of the caudal nTS revealed no change in neuron number but showed a reduced synaptic density in the AD model. Astrocyte number and morphology were unchanged between groups. However, microglia, which are able to influence glutamate‐handling of astrocytes, showed an activated phenotype.

Overall, our data show enhanced glutamatergic signaling in the chemoreflex axis contributing to nTS hyperactivity of the STZ‐AD model. nTS hyperactivity may lead to the observed respiratory dysfunction in this model and potentially constitutes a mechanism for sleep disordered breathing in AD patients.
Gravidity Impacts Respiratory Function, Tracheal Volume, and Jump Performance in Grasshoppers
Authors: Kirkton Scott D. , Valachovic Abigail C. , Zembrzuski Deanna , Raffaelli Daniel , Heiting Grace M. , Sheahan Andrew J.
Published: May 2022

The negative effects of gravidity on respiration and locomotion have been well documented in terrestrial vertebrates but is relatively unknown in insects. During gravidity, the egg mass of Schistocerca americanagrasshoppers can be up to 40% of their body mass. We investigated whether this increase in egg mass impacted oxygen delivery, jump performance, and anaerobic metabolism. Using micro‐dissections and live video analysis, we examined the tracheal system of thirty‐six female grasshoppers across a range of body masses. We found that larger grasshoppers had significantly more eggs, a larger egg mass, and a greater portion of their body dedicated to eggs. In addition, abdominal compressibility (tidal volume) significantly decreased with gravidity. However, there was a significant positive correlation between gravidity and abdominal compression rate, suggesting that gravid grasshoppers make up for smaller tidal volumes by increasing respiratory rate. Furthermore, abdominal compression rates correlated with the inflation of proximal and distal femoral air sacs suggesting a direct impact on oxygen delivery to the jumping muscle. Using microCT analysis, the percent of tracheal volume in the grasshoppers significantly decreased with gravidity (23.6% in nongravid to 13.8% in gravid). During five‐minutes of repeated jumping, gravid grasshoppers had a significantly 30% lower jump rate. However, the average distance per jump did not vary with gravidity. There was no significant effect of gravidity on muscle lactate measured after five minutes of jumping. However, muscle lactate was positively correlated with total distance jumped during the five minutes, which was significantly higher in nongravid grasshoppers. In conclusion, gravidity lowered tracheal volumes, altered ventilatory mechanisms, and reduced jump performance.
Persistent SARS‐CoV‐2 Effects Induce Neuropathy Signature in Dorsal Root Ganglia Underlying Hypersensitivity in a Hamster Model
Authors: Serafini Randal A. , Frere Justin J. , Pryce Kerri D. , Ruiz Anne , tenOever Benjamin , Zachariou Venetia
Published: May 2022

Post‐acute sequelae of COVID‐19, commonly known as long‐COVID, is defined as a persistent symptom(s) that is unexplainable by alternative diagnosis and lasts beyond three months after the onset of COVID‐19. Several studies have now identified long‐COVID in >50% of COVID‐19 patients, emphasizing the need for elucidating mechanisms underlying these symptoms. Pain is a prevalent symptom in long‐COVID patients and presents as headache, persistent muscle pain, joint pain, stomach pain, chest pain, respiratory discomfort, and dysesthesia or paresthesia. Understanding the molecular underpinnings of pain maintenance in these patients could provide information on novel therapeutic strategies. Our lab has previously identified novel treatments for pain due to peripheral inflammation from dysesthesia‐inducing mechanisms associated with acute SARS‐CoV‐2 (SCV2) respiratory infection in hamsters (1‐4 days post‐infection, dpi). We have also previously demonstrated the validity of long‐term infection of hamsters (30‐60 dpi) as a pre‐clinical model of long‐COVID. By utilizing the Von Frey assay, we found that SCV2, but not Influenza A, causes mechanical hypersensitivity at 28 dpi in both male and female hamsters. Bulk RNA sequencing of 31 dpi thoracic dorsal root ganglia (DRGs) revealed a unique transcriptional perturbation signature (168 DEGs, 47 up & 121 down, p‐adj.<0.1), despite viral clearance at approximately 7 dpi. Ingenuity Pathway Analysis of this sequencing (853 DEGs, p‐nom.<0.05) identified several injury‐related canonical pathways, including Production of NO & ROS in Macrophages, Signaling by Rho Family GTPases, mTOR Signaling, Estrogen Receptor Signaling, and Ephrin Receptor Signaling. The Enrichr DisGeNET browser associated these transcriptional changes with clinical neurodegeneration phenotypes, including Amyotrophic Lateral Sclerosis, Alzheimer’s Disease, Neurodegenerative Disorders, and Parkinson Disease. Of note, TUNEL staining did not demonstrate any sign of SCV2‐induced sensory neuron apoptosis at 31 dpi. Further investigation of the sequencing dataset revealed a broad downregulation of TubbmRNA isoforms and Mbp, suggesting microtubule and myelin dysregulation. We also observed a drastic increase in Scn8areads, which could point to Nav1.6‐induced DRG neuron hyperexcitability. In conclusion, our findings suggest that SCV2 leaves a lasting hypersensitivity‐associated transcriptomic signature in DRGs despite early viral clearance that appears to be associated with neurodegeneration mechanisms. As we continue investigating the mechanisms underlying SCV2‐induced actions in DRGs and peripheral nerves, we will also use an upstream regulator analysis of our RNA sequencing data to identify promising therapeutic targets.
Non‐invasively derived human induced mesenchymal stem cells demonstrate an enhanced protection of cardiomyocytes from injuries
Authors: Gurusamy Narasimman , Sigamani Vinoth , Perales Selene G. , Rajasingh Sheeja , Rajasingh Johnson
Published: May 2022

Background

Mesenchymal stem are multipotent stem cells used for treating various diseases including cardiomyopathy. However, their heterogeneity, limited proliferation and low retention in the transplanted tissue restricts their application for cell therapy. Recently, induced pluripotent stem cells (iPSCs)‐derived mesenchymal stem cells (iMSCs) have created an exciting source of cells for autologous therapy.

Objective

In this study, we have compared the therapeutic potential of non‐invasively derived iMSCs with adult umbilical cord MSCs (hereafter referred as MSCs) for the protection of cardiomyocytes from injury.

Methods and Results

The urinary epithelial cells obtained from adult human urine were safely reprogrammed into iPSCs using a mixture of mRNA of pluripotency genes (Oct4, Nanog, Sox2, Klf4, cMyc and Lin28) along with a cocktail of miRNAs. The generated iPSCs were subsequently differentiated into iMSCs, which had the capacity to differentiate into osteocytes, chondrocytes and adipocytes. Our comparative qRT‐PCR and Western blot analysis showed that the mesenchymal‐specific CD73, CD90 and CD105 gene transcripts and proteins were highly expressed in both iMSCs and MSCs. But the iMSCs maintained their mesenchymal markers even at a late passage (P18), during which the MSCs started losing their characteristics. Scratch assay and transwell migration assay demonstrated that iMSCs had a greater wound healing property compared to MSCs. Furthermore, the cardioprotective properties of iMSCs and their exosomes (obtained from the conditioned media by precipitation method) were evaluated using human skin fibroblast‐derived induced‐cardiomyocytes, treated with 50 µg of exosomes 1 hr before subjecting them to either 24 hrs of angiotensin II treatment (10 µM) or 6 hrs of 1% hypoxia and 24 hrs of reoxygenation. In comparison with MSCs, iMSCs and their exosomes elicited an enhanced cardiomyocyte protection studied through measurement of mitochondrial membrane potentials (JC1 dye), intracellular reactive oxygen species (CM‐H2DCFDA), in situ cell death by apoptosis, and qRT‐PCR expression of survival genes. Further studies are underway to evaluate the specific role of exosomal RNA and protein in mediating the cardioprotection.

Conclusion

In this study, we have demonstrated a non‐invasive method of generating iMSCs for regenerative therapy. This homogeneous and highly proliferative iMSCs may provide an alternative source of cells or cell‐free exosomes for treating ischemic cardiomyopathy.
Baroreflex Activation Contributes to the Progression of the Systemic Inflammatory Response in Unanesthetized Rats
Authors: Cesarino Luiz G. , Brognara Fernanda , Castania Jaci A. , Salgado Helio C.
Published: May 2022

Our laboratory showed that electrical activation of the baroreflex could modulate the immune system by controlling the inflammatory response in rats. However, no study has investigated whether physiological reflex activation of the baroreflex also has an anti‐inflammatory effect. Therefore, the objective of the present study was to investigate the effect of physiological activation of the baroreflex in unanesthetized rats, through abdominal aortic coarctation (AAC), on the systemic inflammatory response elicited by lipopolysaccharide (LPS) administration. Sprague‐Dawley male rats (250‐300g) were anesthetized for placing, surgically, a cuff around the abdominal aorta and catheters into the carotid artery and femoral vein. On the next day, the hemodynamic parameters were recorded in the unanesthetized animals. Subsequently, the AAC was carried out for 2 min, followed by the administration of LPS (1.5 mg/kg, i.v.) or saline in the control subjects. After 90 min, blood and spleen samples were collected for subsequent cytokine analysis. As expected, compared to baseline, AAC increased systolic (122 ± 5 vs. 173 ± 10 mmHg), diastolic (90 ± 3 vs. 105 ± 4 mmHg), and mean arterial pressure (106 ± 4 vs. 131 ± 6 mmHg), and reduced heart rate (365 ± 25 vs. 306 ± 17 bpm), confirming the reflex activation of the baroreflex. However, the administration of LPS promoted an increase in heart rate (361 ± 11 vs. 428 ± 21 bpm) over time. Of note, the AAC group associated with LPS administration exhibited, 90 min after LPS administration, a fall in mean arterial pressure (98 ± 7 vs. 88 ± 7 mmHg), combined with an increase in heart rate (362 ± 15 vs. 470 ± 16 bpm). Moreover, compared to the group which received only LPS, the AAC associated with LPS increased the levels of cytokines (IL‐6 and IL‐10) in plasma (IL‐6: 4505 ± 1294 vs. 8782 ± 520 pg/mL; IL‐10: 5 ± 5 vs. 1079 ± 232 pg/mL) and spleen (IL‐6: 3 ± 1 vs. 20 ± 3 pg/mg of tissue; IL‐10: 0.1 ± 0.04 vs. 2 ± 0.4 pg/mg of tissue); potentiating, therefore, the release of cytokines after the induction of the inflammatory process. In conclusion, physiological baroreflex activation elicits a modulatory effect of the immune response during the systemic inflammation elicited by LPS, facilitating the inflammatory process and potentiating the hemodynamic responses promoted by the administration of LPS. Thus, this study showed for the first time that physiological baroreflex activation, through AAC, plays a role in the progression of the inflammatory process.
Digital Body Preservation
Authors: Vandenbossche Vicky , Willaert Wouter , Soltvedt Stian , Smit Noeska , Audenaert Emmanuel
Published: May 2022

Detailed anatomical models can be produced with three‐dimensional (3D) scanning techniques and as such be digitally preserved, archived and subsequently rendered through various media. Here, a novel methodology is presented for combining and matching scan geometry with radiographic imaging. The technique encompasses joining layers of 3D surface scans in an anatomical correct spatial relationship. To do so, a computed tomography (CT) volume is used as template to join and merge different surface scan geometries by means of non‐rigid registration into a single environment. This results in a digital model that can be used in multiple digital learning environments. Finally, as computational expense is usually the main bottleneck in extended 3D applications, the influence of mesh simplification in combination with texture mapping on the quality of 3D models was investigated. The fidelity of the simplified meshes was evaluated in relation to their resolution and with respect to key anatomical features. Large‐ and medium‐scale features were well preserved despite extensive 3D mesh simplification. Subtle fine‐scale features, particular in curved areas demonstrated the major limitation to extensive mesh size reduction. Depending on the local topography, workable mesh sizes ranging from 10% to 3% of the original size could be obtained, making them usable in various learning applications and environments.
Site‐specific regulation of emotional stress responses along the rostrocaudal axis of the insular cortex in rats
Authors: Crestani Carlos C. , Tomeo Rodrigo A. , Gomes‐de‐Souza Lucas , Benini Ricardo , Reis‐Silva Lilian L.
Published: May 2022

The insular cortex (IC) has been described as part of the central network implicated in the behavioral and physiological responses to emotional stress. Besides, it has been reported a site‐specific control of physiological functions along the rostrocaudal axis of the IC. Nevertheless, evaluation of a functional topography of the IC in the regulation of the responses to stressful stimuli has never been documented. Therefore, this study aimed to investigate the impact of acute restraint stress in neuronal activation at different sites along the rostrocaudal axis of the IC in rats. Furthermore, we evaluated the involvement of IC rostrocaudal subregions in the cardiovascular responses to acute restraint stress. For this, male Wistar rats (60‐days‐old) were used. The restraint stress was performed by placing the animals in a cylindrical plastic tube for 60 minutes. For the cardiovascular study, different set of animals had cannula‐guide bilaterally implanted into the anterior, rostral posterior of caudal posterior subregions of the IC; and the non‐selective synaptic inhibitor CoCl2(1mM/100nL) was microinjected 10 min before the restraint onset. We observed that acute restraint stress increased the number of Fos‐immunoreactive cells in the rostral posterior region of the IC (t=2.95, df=14, P=0.0106), while fewer activated cells were identified in the anterior (t=2.44, df=14, P= 0.0276) and caudal posterior (t=2.27, df=14, P=0.0388) regions. Treatment of the anterior region of the IC with CoCl2 did not affect the blood pressure (F(1,15)=0.90, P>0.05) and heart rate (F(1,15)=0.3, P>0.05) increases and the sympathetically‐mediated cutaneous vasoconstriction (F(1,15)=2.23, P>0.05) evoked by restraint stress. However, synaptic ablation of the rostral posterior IC decreased the restraint‐evoked arterial pressure increase (F(1,21)=5.3, P=0.0313), but without affecting the tachycardia (F(1,21)=4.1, P=0.0561) and the reduction in tail skin temperature (F(1,21)=0.05, P>0.05). Besides, bilateral microinjection of CoCl2 into the caudal posterior IC decreased the tachycardia to restraint stress (F(1,17)=11.3, P=0.0036), but without affecting the blood pressure (F(1,17)= 0.2, P>0.05) and tail skin temperature (F(1,17)= 1.4, P>0.05) responses. Taken together, these findings indicate a site‐specific regulation of stress response along the rostrocaudal axis of the IC.
Analgesic and Antinociceptive Effects of (2R,6R)‐hydroxynorketamine (HNK) in Mice
Authors: Yost Jonathan G. , Browne Caroline A. , Lucki Irwin
Published: May 2022

Chronic pain can be challenging to treat and increases the risk of developing psychological disorders such as depression. Identifying novel treatment modalities that effectively alleviate pain is essential to improve clinical treatment and rehabilitation for patients with pain conditions. Ketamine is an effective analgesic for many types of pain. However, its widespread use is limited by its side effect profile and requirement for intranasal/intravenous administration under medical supervision. (2R,6R)‐hydroxynorketamine (HNK) is a ketamine metabolite that lacks the psychotomimetic effects of its parent drug but retains ketamine’s anti‐stress effects. Therefore, it is natural to question whether (2R,6R)‐HNK may also possess analgesic activity.

Administration of (2R,6R)‐HNK produced antinociception in healthy mice exposed to a noxious, painful stimulus 24 hours after injection. The dose response for the delayed‐yet‐persistent antinociception revealed an inverted U shape with significant antinociception at doses of 10‐18 mg/kg for both sexes and 30 mg/kg in female mice. Mice pretreated with different receptor antagonists to examine the potential mechanism for (2R,6R)‐HNK mediated antinociception revealed a mechanism dependent on AMPA receptors and not opioid receptors. In contrast, ketamine antinociception was not dependent on AMPA receptors and partially dependent on opioid receptors. These results demonstrate that both (2R,6R)‐HNK and the parent drug ketamine produce antinociception but work via different neural mechanisms.

In separate studies, (2R,6R)‐HNK administration reversed mechanical allodynia associated with localized inflammatory pain induced in mice by injecting λ‐carrageenan into the hind paw. The onset for this analgesia‐like activity was less than 1 hour with a duration greater than 24 hours following a single administration. (2R,6R)‐HNK was effective at reversing mechanical allodynia at doses of 10 & 30 mg/kg in both male and female animals. These results demonstrate (2R,6R)‐HNK exhibits great promise for treating inflammatory pain in addition to other pain types. Overall, these data suggest that (2R,6R)‐HNK may be a safe alternative therapy for pain that could be made widely available to patients and support the need for continued investigation and development of (2R,6R)‐HNK as a novel non‐opioid pain treatment.
Evaluating Compulsive‐Like Drug‐Taking Using Signaled and Unsignaled Footshock Procedures in Male and Female Rats Self‐Administering MDPV
Authors: Doyle Michelle R. , Peng Lindsey N. , Rice Kenner C. , Collins Gregory T.
Published: May 2022

A subset of rats that self‐administer synthetic cathinones, such as 3,4‐methylenedioxypyrovalerone (MDPV), develop high levels of drug intake and patterns of responding similar to the compulsive, binge‐like patterns of cathinone (e.g., “bath salts”) use reported in humans. Though high‐responder rats exhibit some behaviors thought to be related to substance use disorder, the effects of punished responding in these rats were unknown. These studies evaluated whether (1) high‐responder rats were less sensitive to footshock‐punished responding than low‐responder rats using both unsignaled and signaled footshock procedures; (2) whether sensitivity to footshock was dependent on the self‐administered dose of MDPV. Inhibition curves for unsignaled footshock (0.05‐0.9 mA, increasing across sessions) were evaluated in forty adult Sprague Dawley rats self‐administering 0.01, 0.032, and 0.1 mg/kg/infusion MDPV under a FR5 schedule of reinforcement; footshocks were paired with ~50% of drug infusions. Rats were then trained on a signaled footshock procedure, in which a change in stimulus conditions signaled that the next infusion would be paired with a footshock (intensities individualized to each rat’s IC50). Rats could avoid this footshock by withholding responding for 30‐sec. After rats learned the signaled shock procedure, inhibition curves were generated using a range of footshock intensities (0.05‐0.9 mA). High‐ and low‐responder rats were similarly sensitive to unsignaled punishment by footshock (mean IC50s‐ high‐responders: 0.30mA; low‐responders: 0.36mA). However, high‐responder rats (mean: 45mA) received significantly more total current than low‐responder rats (mean: 18mA). Compared to low‐responder rats, high‐responder rats respond more in the presence of the signal indicating impending shock and avoid a smaller proportion of shocks. This suggests high‐responder rats may be less able to adapt their behavior in response to a stimulus associated with punishment. These studies suggest that the high levels of drug‐taking observed in the high‐responder rats reflects a compulsive‐like pattern of responding, which may be related to continued use despite adverse consequence, a DSM symptom of substance use disorder.
Novel roles for rRNA and ribosome biogenesis associated factors in neural crest cell colonization of the gut and in the pathogenesis of Hirschsprung disease
Authors: Dash Soma , Shannon Stephen , Trainor Paul A.
Published: May 2022

The enteric nervous system regulates the functions of the gastrointestinal system, which include food transportation, digestion, nutrient and liquid absorption and expulsion of waste. The enteric nervous system is comprised of neurons and glia that are primarily derived from a multipotent, migratory population of cells called neural crest cells. Defects in the formation, migration and or differentiation of neural crest cells can lead to incomplete colonization of the gastrointestinal tract and perturbed ENS development resulting in Hirschsprung disease. Hirschsprung disease is a congenital disorder characterized by the absence of ganglia in variable regions of the gastrointestinal tract and ~50% of the known cases of Hirschsprung disease have an unknown genetic diagnosis. This makes it imperative to identify other genes and biological processes that contribute to the etiology and pathogenesis of Hirschsprung disease. We have identified three new genes, Tcof1, Polr1a and Polr1c, and the process of rDNA transcription which is a rate‐limiting step in ribosome biogenesis, as novel regulators of neural crest cell development, enteric nervous system formation and the etiology of Hirschsprung disease. Loss of function mutation of Tcof1, Polr1a and Polr1c in neural crest cells results in total intestinal aganglionosis, the most severe form of Hirschsprung disease in mice. Mechanistically, downregulation of Tcof1, Polr1a and Polr1c results in reduced rRNA synthesis, which leads to increased p53‐dependent NCC apoptosis, and insufficient NCC to colonize the gut. This led us to hypothesize that inhibition of p53 could rescue the Hirschsprung phenotype in the mutants, and we are currently testing this and other and other approaches as potential preventative therapies.
Lipid phosphate phosphatase‐3 dysfunction exaggerates neointimal hyperplasia following diabetic vascular injury
Authors: Subedi Utsab , Bhattarai Susmita , Sharma Sudha , Ara Hosne , murov Milena , Bhuiyan Md Shenuarin , Miriyala Sumitra , Panchatcharam Manikandan
Published: May 2022

Introduction

Diabetes mellitus is a major risk factor for cardiovascular diseases and promotes neointimal hyperplasia characterized by occlusive disorders such as atherosclerosis and stenosis, caused by excessive proliferation and migration of vascular smooth muscle cells (VSMCs) following endovascular interventions such as balloon angioplasty and stent placement. Lipid phosphate phosphatase‐3 (LPP3) is an enzyme that regulates the availability of lysophosphatidic acid (LPA) and has been reported to play a significant role in vascular genesis, cellular permeability, tumor progression, vascular remodeling, and myocardial infarction. However, the functional role and mechanism of LPP3 in diabetic neointimal hyperplasia is unclear. LPP3 dysfunction may lead to LPA accumulation at the vascular injury site, leading to vascular inflammation, proliferation, and migration, causing restenosis. Recently, the Receptor for Advanced Glycation End product (RAGE) has been presented as a potential receptor for LPA leading to downstream signaling in vascular smooth muscle cells (VSMCs).

Methods

In this study, we used a streptozocin‐induced diabetes mice model. We made SM‐22 specific LPP3 knock‐out mice and performed endothelial denudation surgery in the carotid artery to induce neointima hyperplasia to tease out the role of LPP3 in restenosis. We isolated aortic VSMCs from LPP3 floxed and LPP3 knock‐out mice and cultured them under normal or high glucose media. The immunoblotting was performed and probed with antibodies for total extracellular signal‐regulated kinase (ERK) and phosphorylated‐ERK. The cell proliferation and migration assay was performed using the Electric Cell substrate Impedance Sensing (ECIS) with or without LPP3 in VSMC under normal or high glucose conditions. Data are mean ± SEM; P‐values were determined by Student t‐test or one‐way ANOVA with Tukey's post‐hoc tests.

Result

We observed a significant increase in proliferation (P<0.01) and exaggerated migration (p<0.01) of VSMC that lack LPP3 under the hyperglycemic condition in real‐time using ECIS. These effects are at least partially a consequence of decreased lysophosphatidic acid hydrolysis due to lack of LPP3 activity. Furthermore, VSMC that lack LPP3 showed increased phospho‐ERK expression (P<0.01) under hyperglycemic conditions. The diabetic LPP3 knock‐out mice exhibited exaggerated neo‐intimal areas after the carotid injury compared to LPP3 floxed mice.

Conclusion

These findings implicate that LPP3 plays a vital role as a negative regulator of smooth muscle phenotypic modulation. We speculate that changes in LPP3 expression control the development of neo‐intima by limiting smooth muscle cell proliferation, migration, and vascular inflammation in diabetic restenosis.
Variations in the bony architecture of the greater palatine canal: An anatomical report
Authors: Hafeez Sahar , Ansari Ali , Johnson Marjorie , Khadry Galil A.
Published: May 2022

Introduction

The greater palatine canal route has been well‐described in adults for the purposes of anesthetizing the branches of the maxillary division of trigeminal nerve for the relief of sphenopalatine neuralgia [11]. The canal also provides direct access to the contents of the PPF [12]. A complex array of vascular and neural structures characterizes this posterior maxillary region where the GPC is located. Its surgical anatomy can get further complicated by any anatomic variations and identification of vital structures becomes difficult, especially when bleeding during surgery obscures the region. Accurate knowledge of normal anatomy and common anatomical variations therefore remain crucial in minimizing intraoperative and postoperative complications associated with invasive microsurgical approaches to this region. The aim of this study was to explore the architecture of the GPC for any anatomic variations.

Material & Method

In total, 30 adult dried and intact skull specimens were selected. The selection criteria included an intact hard palate with erupted 3rd molars and an intact lateral nasal wall on both sides. The exclusion criteria included any major craniofacial deformities, excessive bone resorption, very old specimens. The bony walls of the GPC were observed by passing a black wire.

Findings

4 out of 30 specimens showed marked variations in the bony medial wall of the greater palatine canal (GPC). Partial to complete malfusion between the posterior surface of the maxilla and the perpendicular plate of palatine bone was observed. This might be the result of embryological malformation.

Conclusion

The present study provided information regarding the existence of some degree of anatomical variation in the bony architecture of greater palatine canal.

Significance

This might help surgeons visualize over the possibility of the existence of any such variation while performing any surgical procedure in the posterior maxillary area. In order to establish the embryological basis of these findings, investigation on a larger number of specimens is highly suggested by the authors.
Cell‐free Hemoglobin‐Oxidized LDL Axis Contributes to Microvascular Endothelial Barrier Dysfunction and Poor Outcomes During Sepsis
Authors: Meegan Jamie E. , Tomasek Toria , Desco Alex , Ware Lorraine B. , Bastarache Julie A.
Published: May 2022

Introduction

Sepsis, a dysregulated host response to infection with high morbidity and mortality, is characterized by a systemic inflammatory response and widespread vascular hyperpermeability leading to edema, organ dysfunction, and death with no specific treatments. Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis‐induced organ dysfunction driven by circulating inflammatory mediators, oxidants, and proteolytic enzymes. Cell‐free hemoglobin (CFH) is one such factor, released from damaged red blood cells during sepsis due to their increased deformability and fragility. Though CFH is not detectable in healthy subjects, plasma CFH is elevated in patients with sepsis and independently associated with mortality. CFH contributes to endothelial dysfunction, but the signaling mechanisms are incompletely understood. CFH can oxidize circulating low‐density lipoproteins (oxLDL), which could potentiate endothelial barrier disruption. We hypothesized that elevated levels of CFH during sepsis are associated with increased oxLDL and contribute to microvascular endothelial barrier dysfunction through the oxLDL receptor, lectin‐like oxidized LDL receptor 1 (LOX‐1).

Methods

Circulating levels of CFH and oxLDL were measured in sepsis patients. In human lung microvascular endothelial cells (HLMVECs), transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell‐substrate Impedance Sensing (ECIS).

Results (Figure)

In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686; p=0.016, n=12) and were higher in patients who died in hospital (33.24 U/L [IQR 28.08, 48.28] vs. 27.19 U/L [IQR 16.24, 38.24]; p=0.045, n=25). HLMVEC barrier dysfunction induced by CFH (p<0.0001, n=6‐7) or oxLDL (p=0.002, n=4‐6) was attenuated by blocking the LOX‐1 receptor with small molecule inhibitor BI‐0115 (Boehringer Ingelheim, 20 µM; p=0.018 vs CFH, n=5; p=0.0002 vs oxLDL, n=6).

Conclusion

Increased plasma CFH and oxLDL are associated with HLMVEC barrier dysfunction and poor clinical outcomes during sepsis; one mechanism by which CFH may cause vascular hyperpermeability during sepsis is through oxidation of LDL which can drive signaling through the LOX‐1 receptor.
Intestinal epithelial CAR‐Like Membrane Protein promotes mucosal barrier function and prevents colitis‐associated cancer in mice
Authors: Anny‐Claude Luissint , Nishio Hikaru , Lerario Antonio M. , Flemming Sven , Hilgarth Roland S. , Watson Sean D. , Zimmerman Zoe , Nusrat Asma , Parkos Charles A.
Published: May 2022

CAR‐Like Membrane Protein (CLMP) is a transmembrane glycoprotein structurally related to Coxsackie and Adenovirus Receptor. Mutations in CLMP gene have been correlated with the development of an intestinal pediatric disorder termed congenital short bowel syndrome (CSBS). To date, not only is the pathogenesis of CSBS incompletely understood, the role of CLMP in gut homeostasis and disease has not yet been elucidated. We found that CLMP expression is significantly reduced in colorectal cancer (CRC) biopsies and increased in inflammatory bowel disease (IBD) compared to normal mucosa samples. Given the expression pattern of CLMP, we generated mice harboring a tamoxifen inducible intestinal epithelial cell (IEC)‐specific deletion of CLMP as well as model human IEC in which CLMP expression was silenced or upregulated. We then assessed the role of CLMP in regulating intestinal mucosal barrier function, the development of chronic inflammation and colitis‐associated cancer. In this study we report that CLMP protein expression is abundant in differentiated IEC compared to undifferentiated and highly proliferative IEC. CLMP is associated with tight junction controlling the paracellular permeability to solutes and macromolecules. Epithelial CLMP deficiency resulted in enhanced paracellular permeability in vivo and in vitro.These findings indicate an important role for CLMP in regulating epithelial barrier function. Mechanistically, we found that CLMP is involved in homotypic trans‐interactions that facilitate cell‐cell cohesion and promotes cell‐matrix adhesion through integrin beta 1 activity. Lastly, we observed that loss of CLMP in human and mouse IEC resulted in increased cell proliferation. Conversely, CLMP overexpression in IEC resulted in reduced cell proliferation. These results were corroborated in murine models of cancer that included xenograft experiments with CLMP‐deficient tumor cells versus CLMP overexpressing tumor cells and Azoxymethane/Dextran Sulfate Sodium‐induced colitis‐associated cancer. In these models, CLMP deficiency worsened tumor growth. Biochemical analyses revealed CLMP‐dependent regulation of IEC proliferation to be mediated through effects on Akt signaling and beta‐catenin transcriptional activity. Collectively, our findings provide the first report of a key role for CLMP in the positive regulation of intestinal epithelial barrier function and prevention of tumor development.
Investigating the Conditionally Essential Role of Human METAP1
Authors: Soens Ross , Cantor Jason
Published: May 2022

CRISPR‐based forward genetic screens make it possible to systematically identify genes required for survival and growth in human cells, and therefore provide a powerful approach to characterize protein function and uncover targetable vulnerabilities in human cancer cells. Notably, it has become appreciated that gene essentiality can be context‐dependent, as CRISPR‐based loss‐of‐function screens across hundreds of human cancer cell lines have uncovered cell‐essential genes that can vary with cell‐intrinsic characteristics such as genotype and lineage. Importantly, environmental factors also influence cell physiology. However, there has been little consideration into how nutrient availability might impact gene essentiality, and most CRISPR‐based screens of human cells are performed in vitro using culture media that poorly recapitulate nutrient conditions in the body. To test the hypothesis that medium composition influences gene essentiality, our lab recently performed genome‐wide CRISPR screens of human blood cancer cell lines in traditional versus Human Plasma‐Like Medium (HPLM), a physiologic medium that we previously developed to more closely reflect the metabolic composition of human blood. Among the strongest HPLM‐essential hits identified from our screen results is METAP1, which encodes one of two human enzymes (METAP1 and METAP2) that can catalyze the cleavage of N‐terminal methionine from nascent polypeptides. Interestingly, these two METAPs are co‐expressed across most human cell lines, are each localized to the cytosol, and also share seemingly similar substrate preferences. However, our screen results indicate that endogenous METAP2 cannot complement the loss of METAP1in HPLM‐cultured cells, suggesting that METAP1 serves a non‐redundant and context‐dependent cell‐essential role. Therefore, we hypothesize that identifying the gene‐nutrient interaction that underlies conditional METAP1essentiality will provide key insights into this unforeseen role. Importantly, we have engineered METAP1‐knockout cells and, in turn, confirmed the conditional CRISPR phenotype for METAP1by performing relative growth assays. Therefore, we are now poised to identify the gene‐nutrient interaction and will proceed to use tools in biochemistry, metabolomics, and molecular biology to explore the context‐dependent role of METAP1 in proliferating human cells.
SIRT6 Protects Against Liver Fibrosis by Negatively Regulating YAP/TAZ
Authors: Chowdhury Kushan , Huang Menghao , Kim Hyeong‐Geug , Dong Xiaocheng C.
Published: May 2022

Hepatic fibrosis often occurs after chronic liver injury, and it is largely attributable to the activation of hepatic stellate cells (HSCs). It is a determining step in the pathophysiology of non‐alcoholic steatohepatitis (NASH), an advanced form of fatty liver disease. The Hippo pathway and its effectors Yes‐associated protein (YAP) and transcriptional coactivator with PDZ‐binding motif (TAZ), have been implicated in hepatic fibrogenesis. However, how YAP and TAZ are regulated in HSCs remains incompletely understood. Here in this work, we have identified Sirtuin 6 (SIRT6), a NAD+ dependent deacetylase, as a regulator of YAP and TAZ in HSCs. Using primary HSCs deficient in SIRT6 from mice fed a moderately‐high‐fat‐cholesterol‐cholate diet (HFCC) for one week as well as in vitro models using transforming growth factor‐β (TGF‐β) treated LX‐2 cells, we have revealed that YAP and TAZ were acetylated during the HSC activation. Moreover, expression of YAP and TAZ target genes including connective tissue growth factor (Ctgf), cysteine‐rich angiogenic inducer 61 (Cyr61) and ankyrin repeat domain 1 (Ankrd1) was increased in those HSCs. Immunofluorescence microscopy analysis of liver sections obtained from Sirt6 HSC‐specific knockout mice fed HFCC diet for six weeks showed elevated YAP nuclear localization in the HSCs, suggesting an activation of YAP during fibrosis. Further in vitroanalysis of YAP/TAZ activity upon gain‐ and loss‐of‐function of SIRT6 in TGF‐β treated LX‐2 cells showed a decrease or increase of Ctgf, Cyr61 and Col3a1gene expression,respectively. To understand how SIRT6 regulates YAP and TAZ, we performed immunoprecipitation and lysine acetylation analysis, and our data showed that SIRT6 indeed interacted with YAP and TAZ and deacetylated them. Taken together, our study has identified a key regulatory axis in which SIRT6 interacts with YAP/TAZ and deacetylates them at several lysine residues, thereby suppressing their transcriptional co‐activator activity during fibrosis.
Directed evolution of single chain antibody library targeting a disintegrin and metalloprotease‐17 (ADAM‐17)
Authors: Raeeszadeh‐Sarmazdeh Maryam , Do Linh
Published: May 2022

Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) are zinc‐dependent metalloproteinases within the metalloproteinase (MP) super family, which play critical roles in remodeling and degrading the extracellular matrix (ECM). Particularly, ADAM‐17’s enzymatic function in the activation or inhibition of cytokines, growth factors and their receptors, and adhesion molecules via cleavage of ectodomains has made this protease a critical target in various inflammatory diseases and cancers. Small molecule therapeutics targeting MPs have caused severe side effects in patients, due to nonspecific binding towards MPs. More specific and selective protein‐based therapeutics, such as antibodies, offer a promising alternative to small molecule therapeutics. Developing a more selective antibody via direct evolution can be labor‐ and time‐intensive. Thus, an algorithm‐based approach, like machine learning, can be utilized to increase mutational analysis, and create a deeper understanding of the correlation between protein sequence and function.

Here, a synthetic single chain antibody (scFv) library that was previously engineered to reduce non‐specific binding, was further engineered via directed evolution, yeast surface display (YSD), and fluorescent‐activated cell sorting (FACS), for improved binding affinity and selectivity to the ADAM‐17 catalytic domain. DNA sequence analysis from each round of FACS indicated that frequent antibody mutations within light and heavy chain complementarity‐determining region 3 (CDR‐L3/CDR‐H3), were responsible for improved binding to ADAM‐17. Improved scFv clones also demonstrated decreased binding affinity towards other MPs. Machine learning was implemented in mutational analysis to further analyze the relationship between sequence and function. The knowledge and tools developed in this research can be used to engineer and design selective enzyme inhibitors. This work will ultimately shed light onto protein sequence‐structure‐function relations using a combination of rational and combinatorial protein design.