bluuebunny/crossref_metadata_2025 huggingface dataset:
Promoting Mitochondrial Biogenesis Protects against Doxorubicin‐Induced Cardiomyocyte Injury
Authors: Uche Nnamdi , Lai Shuping , Dai Qiang , Benjamin Ivor
Published: May 2022

Although anthracyclines such as doxorubicin (DOX) are highly effective chemotherapeutic agents and widely used for various malignancies, they are known to cause dose‐related and life‐threatening cardiotoxicity. This well‐recognized and unpredictable complication of anthracycline therapy may be either permanent and/or reversible, leading to heart failure in many patients while severely impacting quality of life. The pathophysiology has not been fully elucidated but DOX’s activation of cell death pathways and inhibition of mitochondrial biogenesis have been implicated in disease pathogenesis. In recent clinical trials, the β‐blocker carvedilol and the angiotensin converting enzyme inhibitor (ACEi) enalapril have been shown to modestly mitigate cardiovascular events, but the mechanisms remain poorly understood. While prior studies have shown either carvedilol or enalapril might promote mitochondrial biogenesis, we reasoned whether such roles might prevent anthracycline‐induced mitochondrial dysfunction. To test this hypothesis, we investigated rat myoblastic cells (H9c2 cells) and human induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs), which were treated either with DOX or pretreated with carvedilol or enalapril followed by DOX. Mitochondrial respiration was examined using Seahorse assay to evaluate mitochondrial function whereas mitochondrial content was determined via qPCR and flow cytometry. Western blotting and qPCR were also performed to evaluate expression of targets involved in mitochondrial dynamics (fission/fusion) and biogenesis. DOX treatment alone resulted in decreased oxygen consumption rates (OCR) when compared with control. In contrast, carvedilol pretreatment prevented DOX‐induced suppression of mitochondrial function for both H9c2 cells and human iPSC‐CMs. One‐way ANOVA analysis indicated a significant improvement in maximal OCR with carvedilol pretreatment compared with DOX‐treatment in human iPSC‐CMs. We found that DOX treatment expectedly reduced percentage of live cells overall and one‐way ANOVA analysis indicated significant increase in mitochondrial densities. The apparent increased mitochondrial densities in the DOX‐treated group may have resulted from increased fission as opposed to biogenesis. To evaluate this possible association with mitochondrial biogenesis, western blotting and qPCR were also performed to assess for expression of targets involved in mitochondrial dynamics (fission/fusion). Our ongoing studies are evaluating alternative methods to manipulate mitochondrial content as a therapeutic maneuver. Ongoing studies are seeking to refine the combinatorial roles for regulating mitochondrial dynamics as a major contributor for cardioprotection against anthracycline‐induced cardiotoxicity.
Evaluation of the Number of Goblet Cells in Crypt of Colonic Mucosa Submitted to Experimental Gastric Restriction
Authors: Akamatsu Flávia E. , Nunes Camila , Fontes Luiz G. , Itezerote Ana M. , Saleh Samir O. , Hojaij Flávio , Andrade Mauro , Pereira José A. , Martinez Carlos A. R. , Jacomo Alfredo L.
Published: May 2022

Obesity‐related comorbidity is defined as a condition caused by overweight/obesity or known to contribute to the presence or severity of the condition. These comorbid conditions are expected to improve or go into remission in the presence of effective and sustained weight loss. However, the mechanisms of surgical weight loss and the long‐term consequences of bariatric surgery remain unclear. World Health Organization reports obesity as a rising chronic condition with many devastating health consequences. Gastric reduction procedures are currently employed to treat morbid obesity and there are few reports concerning the consequent effects in large intestine.

Methods

Experimental model of gastric restriction was devised using rats. The animals were submitted to surgical gastrostomy and a cylindrical loofah was inserted into the stomach. We studied 30 adult male Wistar rats divided in three groups: the stomach reduction group (R20), the sham group (SH20), which underwent the same procedure except for the loofah insertion and the control group (C20). Animals were fed and kept in separate cages. Two colon samples were collected 2 cm from the cecum and were individually packed into containers. Samples were submitted to routine histological processing for Alcian Blue, Periodic Acid and Reagent of Schiff (PAS) and High Iron Diamine (HID AB). The sections were analyzed for the length of the crypts and mucous cells. Goblet cells were obtained in all groups as mean, median, minimum, and maximum and were compared between groups using generalized estimation equations (GEE) with normal distribution and identity link function supposing exchangeable correlation matrix between fields. Bonferroni's multiple comparison was applied to identify differences between groups. The results are described by the mean value with respective standard deviation.

Results

The analysis of PAS and AB Goblet cells count on R20 group indicated a statistically significant reduction and no shallower crypts as compared to the C20. The sialomucins and sulphomucins number cells were found with no enhance. There was not difference regarding on R20 compared to C20 and SH20.

Conclusion

These findings might be a result of colonic mucosae atrophy secondary to the effects of gastric restriction in this model. Restriction decreases the content of neutral mucins and total acidic mucins, without interfering with the sulfomucin and sialomucin acid mucin subtypes.
Effect of Zinc in Submandibular Salivary Gland Branching Morphogenesis
Authors: Kazi Gulsan Ara Sathi
Published: May 2022

Introduction

Nerves from the parasympathetic branch are critical for the function of the glandular tissue including salivary gland. Parasympathetic innervation influences organ regeneration: tissue regrowth impaired after parasympathectomy in the salivary gland. As it is difficult to treat the damaged nerves, there is a need to develop new materials and treatment methods to treat the nerves. Zinc ions are reported to be important metal ions in the physiological functions of the nervous system and involved in neural cell proliferation and differentiation. Here, we aimed to study the influence and neuroprotective role of the extracellular zinc during submandibular salivary gland (SMG) development using ex vivo tissue culture system. Also we prepared zinc ion‐containing agarose hydrogel beads (Zn+Aga) for sustained release of zinc ion.

Materials and Methods

300~500µm size beads prepared. Different concentration of zinc were incorporated in the beads. LY294002 an antagonist of PI3Kinase pathway also incorporate as needed. Next, 0.3 wt%~4 wt% alginate hydrogel sheets prepared for ex‐vivo organ culture. SMG were isolated from the pregnant ICR mouse at day 13 (E13) and culture on hydrogel sheet with or without Zn ion and LY294002 containing beads. Finally, the data analyzed histomorphometrically.

Results

We successfully prepare the Zn+Aga and observed the sustain release of Zn+ upto 48 hours. Histological evaluation showed that zinc ion promoted the growth of submandibular tissue. The immuno expression of AKT was promoted by the administration of zinc ion. AKT decreased by using ly294002, which is an inhibitor of PI3kinase, and it recovered after administration of zinc. This indicates that zinc administration is involved in the AKT‐pathway.

Discussion & Conclusion

Collectively, these data clearly illustrate that the Zn is essential element during salivary gland organogenesis through activation of the PI3Kinase‐AKT pathway.
Role of Adhesion Mediator SibA in Mechanotransduction of Dictyostelium discoideum
Authors: Perez Alexia , Artemenko Yulia
Published: May 2022

Dictyostelium discoideum is a social amoeba commonly used as a model organism for the study of various cellular processes, including directed cell migration. Mechanical cues, such as shear flow, have the ability to induce directed migration. However, little is known about the role of cell‐substrate adhesion in mechanotransduction of D. discoideum. We hypothesized that adhesion is essential for the ability of cells to sense and respond to mechanical cues. To test this hypothesis, we examined whether mechanotransduction depends on SibA, a transmembrane protein involved in the adhesion of D. discoideum. Before testing cell response to shear flow, we studied the adhesion of SibA‐null cells grown under our experimental conditions. Adhesion of SibA‐null cells in growth media was significantly reduced compared to wild‐type cells, consistent with previously published findings. Notably, adhesion was significantly decreased by 25% in SibA‐null compared to wild‐type cells grown on a bacterial lawn, which is the growth method used prior to the assessment of response to shear flow. To test how SibA‐null cells respond to acute mechanical stimulation, we examined the localization of active Ras as a read‐out of the activation of the signal transduction network involved in directed migration. Both wild‐type and SibA‐null cells had a comparable response to 2‐second stimulation with shear flow at two different pressures. These findings suggest that the modest reduction in adhesion observed in SibA‐null cells did not affect the ability of cells to respond to shear flow stimulation. Whether more substantial changes in adhesion would perturb mechanotransduction in this system remains to be examined.
Evidence for a Highly Cytokine‐Sensitive Network of Iron‐Associated Genes in Islets
Authors: Counts Grace P. , Slepchenko Kira G. , Sharma Poonam R. , Corbin Kathryn L. , Qureshi Farhan M. , Colvin Robert A. , Nunemaker Craig S.
Published: May 2022

Among the complex factors contributing to beta‐cell failure is inflammation. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)‐1beta and IL‐6, increases the risk of developing type‐2 diabetes (T2D), and there is evidence that these low levels of circulating cytokines lead to islet dysfunction. Iron is closely connected to both the inflammatory response and diabetes. High levels of dietary iron increase risk of developing T2D, and excessive iron uptake by beta‐cells can cause oxidative stress and inhibit function. In this study, islets were treated for 48h with 10 pg/mL IL‐1beta + 20 pg/mL IL‐6 as a model of low‐grade inflammation. Analysis of gene microarray data identified three iron‐associated genes among the most cytokine‐sensitive: HAMP, STEAP4, and LCN2. These proteins are all involved directly or indirectly with increasing and/or sequestering cellular iron. RT‐PCR following exposure to various stressors known to induce beta‐cell failure revealed upregulation of HAMP, STEAP4, and LCN2 to be cytokine‐specific. Overexpression of STEAP4 induced upregulation of HAMP and LCN2, while ferritin and ferroportin expression were unaffected. Beta‐cells scanned by synchrotron X‐Ray fluorescence provided data showing cytokine exposure alters iron distribution. Iron was found in discrete structures (area ~0.15‐0.45 µm2) that were significantly smaller and more iron‐dense in cytokine‐treated beta‐cells, consistent with the sequestering function of identified iron‐associated genes. These data suggest a network of iron‐regulating genes in beta‐cells plays a role in sequestering iron in response to low‐grade inflammation.
Assessing High‐ and Low‐intensity Physical Rehabilitation Approaches to Address Skeletal Muscle Dysfunction Following Volumetric Muscle Loss
Authors: Schifino Albino G. , Heo Jun‐Won , McFaline‐Figueroa Jennifer , Greising Sarah M. , Call Jarrod A.
Published: May 2022

INTRODUCTION

Volumetric muscle loss (VML), the removal of a large volume of muscle tissue due to surgery or severe trauma is characterized by persistent contractile and metabolic deficits. There is a need to identify clinically translatable physical rehabilitation approaches to address VML; however, optimal parameters of physical rehabilitation strategies such as intensity, progression, and volume have not been well‐established. The primary objective of this study was to determine if the prescribed intensity of two clinically translatable rehabilitation strategies, i.e., whole‐body vibration and voluntary wheel running, influenced skeletal muscle mass and function after VML. We hypothesized that physical rehabilitation would improve muscle function versus leaving the injury untreated, and that magnitude of improvement would be influenced by intensity of physical rehabilitation. METHODS: 12‐week‐old, male C57BL/6 mice (N=55) were assigned to one of 5 groups: uninjured, VML with no rehabilitation (VML), high‐resistance wheel running (HR), low‐resistance wheel running (LR), high‐magnitude vibration (H‐Vib, 1.0g), and low‐magnitude vibration (L‐Vib, 0.6g). Mice underwent unilateral VML injury to the primary ankle plantarflexors (gastrocnemius, soleus, plantaris muscles). Physical rehabilitation strategies were administered for 8‐weeks starting 3‐days after VML injury. To determine rehabilitation effectiveness, muscle mass, muscle strength (i.e., in vivopeak‐isometric torque), and muscle metabolic function (permeabilized muscle fibers oxygen consumption and electron conductance) were assessed. RESULTS: Compared to uninjured mice, VML without rehabilitation resulted in 27% less muscle mass (p<0.01), 36% less peak‐isometric torque (p<0.01), 22% less maximal oxygen consumption (p<0.01) and 36% less electron conductance (p<0.01). Vibration: There were no statistically significant differences among groups for muscle mass or mitochondrial function (p≥0.50). Peak‐isometric torque was 24% greater in H‐Vib (p<0.01), but not L‐Vib mice, compared to VML. Wheel running:HR, but not LR, had 11% greater muscle mass compared to VML (p<0.05). Peak‐isometric torque was 22% greater in both HR and LR compared to VML (p<0.05). Metabolic function was not statistically different among groups. CONCLUSION: Our primary finding is that vibration and wheel running are capable of producing positive adaptations in VML‐injured muscle. Furthermore, results from this study suggest that intensity as a rehabilitation variable should be considered when developing and validating strategies to address functional deficits in VML‐injured muscle. Ongoing and future research is needed to determine the molecular signaling pathways affected by these rehabilitation strategies, and more importantly, why metabolic adaptations are lacking.
Leucocytes are Mass‐Invariant in Reptiles Unlike in Endotherms
Authors: Fletcher Julie E. , Martin Lynn B. , Downs Cynthia J.
Published: May 2022

How do large and small animals defend against parasite challenges given the consequences of body mass on physiology and disease transmission? Functionally equivalent mammalian and avian granulocytes increased disproportionately with mass (i.e., hypermetrically), such that large organisms have higher concentrations than expected by mass alone. However, how ectothermic leucocyte concentrations scale is understudied. We hypothesized that differences exist in the scaling of leucocyte concentrations between endotherms and ectotherms as these groups have substantial differences in their physiology and immunology. Analysis was conducted using MCMCglmm, phylogenetically informed models to determine which existing scaling hypothesis best predicted relationships between mass and lymphocyte and heterophil concentrations among >120 species of reptiles and compared our results with those from birds and mammals. We also compared body mass and life history as a predictor of variation in immune cell concentrations. Reptilian lymphocyte concentrations scaled close to isometrically (i.e., mass‐invariant; phylogenetic MCMCglmm: b(mean, 95% CI) = ‐0.040, [‐0.072, ‐0.007]), and the slope was indistinguishable from the slope found for mammals and birds. In contrast, reptilian heterophil concentrations scaled isometrically (b =0.006, [‐0.028, 0.040]), whereas functionally equivalent mammalian and avian granulocytes scaled hypermetrically. Although reptilian leucocytes were mass‐invariant, the slope was greater than predicted if metabolic rate serves as a pace marker for immune defenses, suggesting that large organisms have greater protection than expected by the Rate of Metabolism Hypothesis. Life history was also a poor predictor of variation in reptilian heterophil and lymphocyte concentrations. Our results provide novel insight into large scale patterns of immune defenses of endothermic and ectothermic vertebrates and have direct implications for modeling the evolution of immune defenses and identifying parasite reservoirs.
Targeting p21‐highly‐expressing Senescent Cells Enhances Skeletal Muscle Function through Mitochondrial Function and Reactive Oxygen Species
Authors: Noh Sung Gi , Ahn Ahram , Wang Lichao , Xu Ming , Kwon Oh Sung
Published: May 2022

BACKGROUND

The accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria‐derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet‐induced muscle dysfunction.

METHODS

Five p21‐Cre/+; +/+ (P) and p21‐Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria‐derive ROS production was determined using Amplex Red assays. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P < 0.05).

RESULTS

Reductions in complex I + II state 3 respiration were observed in p21‐Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s‐1·mg‐1; Obese P: 17.45 ± 2.85 pmol·s‐1·mg‐1; Obese PD: 30.04 ± 3.19 pmol·s‐1·mg‐1; P < 0.05). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P<0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s‐1·mg‐1; Obese P: 26.83 ± 0.54 pmol·s‐1·mg‐1; Obese PD: 12.23 ± 0.54 pmol·s‐1·mg‐1; P<0.05).

CONCLUSION

These results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria‐derived ROS and enhancing mitochondrial function.
An Insight into the Role of Mesothelin in Pediatric Acute Myeloid Leukemia
Authors: Faust Joshua R. , Hamill Darcy , Kolb E. A. , Gopalakrishnapillai Anilkumar , Barwe Sonali P.
Published: May 2022

Pediatric acute myeloid leukemia (AML) is characterized by the malignant transformation of hematopoietic stem and progenitor cells that lead to the accumulation of immature myeloid cells in the bone marrow. Standard treatment of AML includes intense cytotoxic chemotherapy and hematopoietic stem cell transplants with relatively low cure rates. Targeted therapy options for AML are limited. Mesothelin (MSLN) is a 40‐kDa glycosylphosphatidylinositol (GPI)‐anchored protein that is highly expressed on many tumors, but lacks expression in many normal tissues, making it an excellent target for cancer therapies. More recently, the identification of MSLN expression in a subset (about one‐third) of pediatric AML patients warranted preclinical studies with MSLN‐targeted therapies, including immunotherapy. Recently, our lab utilized bispecific T cell engaging antibodies to recruit T cells to MSLN‐expressing leukemic cells, increasing in vivo survival rates in patient‐derived xenograft models.

MSLN has been shown to play a role in promoting proliferation and invasion in mesothelioma, ovarian cancer, and pancreatic adenocarcinoma, mainly through interactions with MUC16/CA125. Current investigative efforts in AML focus on MSLN‐targeted therapies, while the function of MSLN in AML remains unresolved. Previously, we identified a correlation between MSLN expression and extramedullary disease, as well as enhanced leukemic burden in xenograft models. Here, we report that MSLN expression is associated with increased rates of cell proliferation and migration.

The proliferation rate of MSLN‐negative AML cell lines, MV4;11 & NOMO‐1‐KO, and two MSLN‐expressing cell lines, MV4;11‐MSLN & NOMO‐1, was evaluated by BrDU proliferation assay or direct measurement of cell numbers over 96 hours. MSLN‐expressing cells proliferated at an increased rate comparative to the non‐expressing cells. Consistent with higher proliferation rate in MSLN‐expressing cells, these cells showed reduced percentage of G1 population and increased population in the S phase of the cell cycle. In a Transwell migration assay, higher number of MV4;11‐MSLN cells migrated towards serum compared to their wild‐type counterpart, indicating that MSLN expression promoted cell migration. Our data suggest that MSLN expression in pediatric AML cells enhances proliferation and migration. Therefore, targeting MSLN may be beneficial to curb AML progression.
Use of the Lipolysis Inhibitor Acipimox Fails to Modulate Chronic Muscle Protein Synthesis Rates or Attenuate the Loss of Muscle Mass in a Rodent Model of Severe Burns
Authors: Vardarli Emre , Bhattarai Nisha , Abdelrahman Doaa , El‐Ayadi Amina , Murton Andrew
Published: May 2022

Objective

Massive burns result in chronic lipolysis of adipose stores resulting in ectopic lipid accumulation. Given reports that hyperlipidemia and/or increased intramyocellular lipid accumulation is associated with impaired synthesis of muscle proteins, we hypothesized that administering the lipolysis inhibitor acipimox could protect muscle protein synthesis capacity in a rodent model of burns, thereby blunting the loss of muscle mass.

Methods

Sprague Dawley rats (4 groups, 16 animals per group, n = 64) were randomized to receive a scald burn (~ 60 % total body surface area; n=32) or sham procedure. Animals in each group were assigned to receive either vehicle (saline) or acipimox (50 mg/kg/day) daily by i.p. injection for 7 days. All animals received a bolus of deuterated water (10 ml/kg, i.p.) followed by supplementation in drinking water (2 %) to monitor habitual muscle protein synthesis rates (MPS). On day 7, animals were sacrificed, and tissues collected. MPS was calculated by measuring intracellular and bound deuterium enrichment of alanine via Gas Chromatography Isotope Ratio Mass Spectrometry.

Results

Severe burns resulted in significant body weight loss compared to the sham injury group (7 % vs 10 %; p < 0.001). However, acipimox administration did not prevent the loss of body weight. There was a trend for gastrocnemius and tibialis anterior muscle weights to be lower in burn animals (P = 0.058 and P=0.057, respectively). However, acipimox did not prevent the loss of muscle weight with burn injury. Neither burn injury nor acipimox administration impacted habitual MPS rates.

Conclusion

In sharp contrast to what is observed in patients, severe burns do not alter habitual MPS rates in rats. Given evidence that rats still experience a loss of muscle mass in response to thermal injury, it suggests that the dominant mechanism by which muscle protein content declines in these animals is via enhanced muscle proteolysis. Acipimox appears to exert no beneficial impact on muscle protein homeostasis in burn injured rodents.
Progranulin as a Context Dependent Signaling Molecule
Authors: Okonya Chika , Pascual Agnes , Uppalapati Chandana , Leyva Kathryn , Hull Elizabeth
Published: May 2022

Progranulin is a signaling molecule which has roles in lysosomal function, neurodegenerative diseases, wound healing, inflammation, and proliferation. The progranulin protein is composed of 7½ highly conserved granulin repeats joined by linker regions which are subject to proteolytic processing. Thus, the ultimate biological activity of progranulin is context dependent as it relies on the relative expression of diverse intracellular and extracellular proteases and protease inhibitors. The SW13 human adrenocortical carcinoma line exists in two epigenetically distinct subtypes which both express progranulin and is therefore a uniquely powerful model in which to probe the relationship between progranulin processing and cellular activity. Treatment with low doses of exogenous progranulin increases growth of SW13‐ but not SW13+ cells. However, SW13+ cells increase their growth rate at higher progranulin doses. Together, these data are consistent with an inability of SW13+ to process endogenous progranulin to the mitogenic granulin unit(s). This conclusion is supported by immunoblotting with antibodies specific for granulin repeats revealing that progranulin is processed differently in each subtype. In this work, we focused on lysosomes as a potential site for differential processing of progranulin as lysosomal proteases have been shown to process progranulin and progranulin itself plays a role in lysosome maturation. Differences in lysosomal pH and function between the two subtypes have been detected using two pH‐sensitive dyes which localize to the lysosome. Ongoing experiments are aimed at probing the ability of differential processing of progranulin to influence which subtype the cell line adopts.
Cyclin C's Role in Myoblast Differentiation‐Induced Mitochondrial Fragmentation
Authors: Campbell Alicia , Strich Randy
Published: May 2022

One of the largest and most dynamic tissues in the body, skeletal muscle, requires constant regeneration and upkeep. Dysregulation of this regeneration process has been implicated in many neuromuscular diseases and myotonic dystrophies. Regeneration requires the differentiation of myogenic lineages including exiting the cell cycle, gene expression changes, and fusing of myoblasts into multinucleate myotubes. Part of this reconstruction requires the breakdown and repopulation of mitochondrial networks. At the early onset of myoblast differentiation, there is an upregulation of dynamin‐related protein, Drp1, and an increase in mitophagy mediated by sequestosome (SQSTM1) removal of mitochondria. Previously, our lab has shown that mitochondrial fragmentation following stress requires the transcriptional regulator cyclin C, the regulatory subunit for cyclin‐dependent kinase 8 (Cdk8). Preliminary data indicate that cyclin C is required for mitochondrial fragmentation during myoblast differentiation. At the early onset, cyclin C co‐localizes with the mitochondria, as visualized with indirect immunofluorescence. Cells were additionally treated with PFTμ, a cytosolic chaperone inhibitor that blocks translocation of cyclin C to the mitochondria, and in turn inhibits cyclin C‐mediated mitochondrial fragmentation. This treatment resulted in lack of mitochondrial fragmentation typically seen during the differentiation process. In addition, efficiency of differentiation was quantified using gene expression of myogenic regulatory factors (MRFs) MyoD and Myosin Heavy Chain (MyHC), which are normally expressed in a temporal manner throughout differentiation. Treatment of cells with PFTμ significantly decreased expression of MyoD by 1.2 fold compared to endogenous controls, as well as a 1.6 fold decrease in MyHC expression. Future studies aim to elucidate the role of cyclin C‐mediated mitochondrial fragmentation during myoblast differentiation and how this relates to the homeostasis of muscle regeneration processes.
Candidate diagnostic and prognostic markers for pancreatitis and pancreatic ductal adenocarcinoma
Authors: Wilkie Thomas M. , Kantheti Havish
Published: May 2022

Diagnostic markers are desperately needed for early detection of pancreatic ductal adenocarcinoma (PDA). We describe sets of markers expressed in temporal order in mouse models during pancreatitis, PDA initiation and progression. Cell type specificity of PDA marker expression was analyzed by interrogating quantitative dot plots of single cell (sc) RNAseq for 3021 candidate genes in tumor samples from a mouse model for PDA (KIC) at early and late stages of PDA progression compared to normal pancreas. Protein markers were identified by differential mass spectrometry (MS) of mouse tumors and conditioned media from human cancer cell lines. Additional candidate genes were identified from signaling pathways that drive PDA, including the Ras pathway, calcium signaling, and known cancer genes. Developmental progression of cancer was revealed in seven classes of PDA markers expressed in cancer cells and stroma. The earliest diagnostic markers (47 genes) were specifically expressed in epithelial cancer cells in early, but not late, PDA tumors. Other markers were first elevated in epithelial cancer cells of late stage tumors (288 genes), or in both epithelial and mesenchymal cells (740 genes), or only in mesenchymal cells (216 genes). Stromal markers were differentially expressed in early‐ and/or late‐PDA neoplasia (594 genes). Pancreatitis is a risk factor for PDA in humans. Mouse models of pancreatitis, including caerulein treatment and acinar‐specific homozygous deletion of differentiation transcription factors (dTFs), were screened for early expression of all PDA markers identified in KIC neoplasia. Select diagnostic markers were validated by immunohistochemistry in mouse and human samples of normal pancreas, early‑ and late‐stage PDA. Prognostic markers from TCGA were identified that showed differential and cell type‐specific expression in PDA. Here we present diagnostic and prognostic markers for disease progression from pancreatitis to late‐stage PDA.
Voltage Stimulation of the Anterior Ethmoidal Nerve in Rats Produces a Cardiorespiratory Response Similar to the Diving Response
Authors: McCulloch Paul , Silvis Clarence , DiNovo Karyn
Published: May 2022

The diving response, initiated when animals submerge under water, includes a decrease in heart rate, peripheral vasoconstriction, and apnea. Electrical stimulation of the anterior ethmoidal nerve (AEN), the nerve that innervates the nasal passages, can produce similar reflex responses. The purpose of the present research in Sprague‐Dawley rats was to electrically stimulate the AEN using silver coated copper wire and a voltage stimulus supplied through an iWorx TA‐220 Control Module and Labscribe software. Our hypothesis was that increasing the intensity of the AEN electrical stimulation would increase the intensity of the resultant cardiorespiratory responses. Accordingly, we isolated the AEN at the base of the orbit, hooked the electrodes around the nerve, and insulated the nerve and orbit with dental impression material. While recording heart rate, arterial blood pressure and respiration, we varied 5 s of AEN electrical stimulation between 0.1 and 1.1 volts. Stimulation threshold of 0.2 volts would elicit slight bradycardia, slight alterations in BPa, and a slight decrease in respiratory rate. With increasing voltages, the response intensified. When maximally stimulated, HR decreased from 438 ± 28 to 181 ± 37 bpm, and BPa decreased from 127 ± 5 to 121 ± 14 mmHg. The respiratory rate decreased from 93 ± 3 to 0 breaths/min with an apnea duration of 5.0 ± 0.2 s. Our studies found that the intensity of the cardiorespiratory responses and the voltage of the AEN stimulation varied somewhat linearly. This technique and these findings can be used in future research to investigate the brainstem pathways of the mammalian diving response.
Epigenetic Analysis of Different Tissues using Oxford Nanopore Technology
Authors: McEvoy Jack , Ecker Geoffrey
Published: May 2022

Global changes in the epigenetic landscape are hallmarks of cancer and many other diseases. One component of cancer epigenetics is DNA methylation. DNA methylation is responsible for regular biological processes, but decreased methylation leads to changes in gene expression which leads to an increase in cancerous activity. Methylation analysis allows insights into gene regulation and disease predispositions. Many different technologies are able to analyze DNA methylation through DNA/RNA sequencing. Nanopore Technology is used to analyze, in real time, long nucleotide fragments. The technology works by monitoring changes in electrical current through the membrane within the flow cell. Tissue samples were collected, and DNA was extracted using the Monarch Genomic DNA Purification Kit. Nanopore’s Rapid Sequencing Kit/Protocol was used to carry out the DNA extraction steps before sequencing. Real time DNA sequencing was conducted using Nanopore’s MINION and Flongle Cell. Epigenetic analysis was performed using secondary softwares Megalodon, Guppy basecaller, and EPI2ME. Studying epigenetic environments can furtherer understanding of disease etiology and understanding the function of diagnostic biomarkers.
Amalgamation of Arts, Anatomy and Passion in Creating an Anatomy Atlas for Active Learning
Authors: Mukhopadhyay Soma
Published: May 2022

The objective of this study was to develop a photographic atlas of rabbit anatomy to provide a low cost and effective learning tool to help students understand anatomy better.

Hypothesis

The newly developed interactive anatomy e‐atlas will provide a better learning resource and reference for Gen Z students and help them to succeed.

A brief statement of methods:

System Approach – Selection of organ system

Development of hand drawn maps and photographic plates

Collection of feedback from Teaching Assistants (TA) and peers

Improvement and preparation of additional photo plates

Establish collaboration with instructional division to get additional help. Collaboration with peers

Use in class and IRB submission

Continue using the resource, collection of feedback.

Take trainings to improve photography skills.

Modifications and additions based on the feedback.

A summary of the results

Data collected so far shows a clear positive trend expressing effectiveness and usefulness of the newly created anatomy resource for muscular and vascular system of rabbit.

A statement of the conclusions.

In conclusion it could be said that the newly developed resource (Photographic Atlas of Rabbit Anatomy) has become an important and necessary tool for student learning. Collecting data will be continued for next three years to monitor its effectiveness in helping students to develop the skill for dissection and identification and gaining knowledge about anatomical distribution of muscles and blood vessels.
Neuronal Nitric Oxide Synthase and Neural growth factor interaction in the development of ventricular hypertrophy toward heart failure in the spontaneously hypertensive rats
Authors: Bocanegra Alfaro Ana , Navarrete Peñaloza Grecia , Cortes Gallardo Ernesto , Gómez Ma de Jesus
Published: May 2022

Ventricular hypertrophy (VH) is associated with an increased expression of neural growth factor (NGF) in spontaneously hypertension rats (SHR). In vitro studies suggest a strong interaction between NGF and neuronal nitric oxide synthase (nNOS), then in this study was determined the probable interaction between nNOS and NGF in the development of ventricular hypertrophy and heart failure in the SHR strain. Male Wistar Kyoto (WKY) and SHR strain at 2 and 8 months old were studied, and in accord with heart and ventricular weights and the presence of ventricular dilatation, SHR was classified as ventricular hypertrophy (SHR‐VH) and heart failure (SHR‐HF). Heart rate (HR), systolic arterial blood pressure (SABP), body, heart and ventricular weights, were analyzed. nNOS and NGF levels were measured in LV homogenates through Elisa assays, also nitrites levels in left ventricular homogenates were measured through Griess reaction in the experimental groups. The results show significantly differences between WKY and SHR strain in the parameters measured at 2 and 8 months old. HR, heart weight was significantly higher in SHR‐HF in comparison with WKY at the two edges studied. SABP was significantly different between WKY and SHRs, only at 8 months old. NGF in ng/ml, was increased in SHR‐LV at 2 and 8 months old (2.3±0.011, 2.6±0.017) whereas was reduced in SHR‐HF (1.43±0.015, 1.26±0.011) in comparison with the WKY (2.23±0.018, 1.89±0.015) P<0.05. The same pattern was observed with the nNOS levels in the experimental groups and was significantly different at 8 months old between experimental groups (nNOS in U/L; SHR‐VH‐64±6.9, SHR‐HF: 41±5 versus 54±3.5 in WKR) P<0.05. LV nitrites mean level in µM/L show a significantly increased in the experimental groups at 8 months old in comparison with the group at 2 months old (14.55±1.3 at 8 months against 4.3±0.88 at 2 months old, P<0.05). In conclusion the results shows that nNOS and NGF follows a similar pattern in the cardiac hypertrophy and heart failure, suggesting a positive interaction between them and that the preservation of nNOS and NGF production is associated to compensated ventricular hypertrophy, whereas reduced of them is associated to heart failure.
Analysis of epidemiology and novel mutation of Helicobacter pylori antibiotic resistance in South Korea
Authors: SoonYoung Park
Published: May 2022

Background

Helicobacter pylori is a Gram‐negative bacterium associated with upper gastrointestinal disease, peptic ulcer, mucosa‐associated lymphoid tissue lymphoma and eradication treatment is recommended because it is one of the causes of gastric cancer. However, resistance to some antibiotics used for treatment has developed, the eradication rate is declining worldwide. This study focused on antibiotic susceptibility test (AST) of H. pylori isolated from patient clinical specimens. The aim of this study is to identify the resistance epidemiology of H. pylori and to elucidate the resistance mechanism through gene analysis.

methods

H. pylori isolated from patient clinical specimens is identified and AST for amoxicillin (AMX) and clarithromycin (CLR) are performed, and the results are analyzed through Whole Genome Sequencing (WGS) and MultiLocus Sequence Typing (MLST), respectively. The strain type of the MLST result is compared with the CLR resistance minimum inhibitory concentration (MIC) value to analyze the significant points.

H. pylori is cultured on EYE Agar plate and incubated in Anaerobic Jar for 2~5 days with microaerophilic air condition (5% O2, 10% CO2, 85% N2). Cultured isolates are identified by MALDI‐TOF system and performed AST using Epsilometer test to determining MIC.

Results

366 H. pylori were isolated from 1952 clinical patients, and 36 AMX‐resistant H. pylori and 101 CLR‐resistant H. pylori were identified. Among the identified H. pylori, 9 AMX high MIC strains were selected and WGS was performed, and 12 CLR high MIC strains were selected and MLST was performed.

As the WGS result of 9 clinical strains resistant to AMX, single nucleotide polymorphism (SNP) of the potential target gene was analyzed with reference to ATCC 26695. Six genes, Penicillin binding protein1A (PBP1), PBP2, NhaC, hofH, hofC, and hefC, were selected as potential target genes. As a result of the analysis, 8, 7, 4, 9, 16, and 2 SNPs were found, respectively, and it was specifically confirmed that there were many SNPs in the outer membrane component Gene. However, No correlation was found between AMX MIC value and target SNP.

12 For the CLR resistance strain, MLST was performed using 7 housekeeping genes (atpA, efp, trpC, ppa, mutY, yphC, Ure1). In 12 strain, 10 CLR‐resistance H. pylori are not identified strain type(ST) to The National Center for Biotechnology Information(NCBI) database because they have a lots of mutation in 7 housekeeping gene. 2 CLR‐resistance H. pylori are identified the new type strains.

Conclusion

In this study, we conclude that hofC, hofH related to outer membrane protein component may be associated to the AMX resistance. And additional research is continued to need about other resistance potential gene, especially hopC.
Epigenetic Age Acceleration as a Risk Predictor of Hepatocellular Carcinoma
Authors: Luna Aesis , Thrush Kyra
Published: May 2022

Introduction

Mortality rates from hepatocellular carcinoma (HCC) have been steadily increasing in the US over the past 15 years. Most patients are diagnosed at advanced stages when the mortality rate is as high as 89%. Early diagnostic indicators are not currently available, and unfortunately, invasive liver core biopsies or resections are the standard of care to confirm HCC diagnosis. While no mutations have have proven to be tumor drivers, novel genome wide methylation studies show clear, cancer‐specific patterns of methylation and identified deregulation of epigenetic signatures. Epigenetic clocks have been shown to be a versatile tool for the prediction of a variety of patient phenotypes. Most commonly used for the prediction of age and mortality risk, these have also been used as indicators of alcohol consumption, smoking, and proxies for various blood‐based biomarkers. Acceleration in a number of clocks has been correlated to increased all‐cause mortality, as well as cancer risk. While much focus in HCC has been on clocks trained to precisely predict chronological age, confidence in the characterization of the notable epigenetic landscape and target region identification will be improved through the use of clocks trained to instead predict biological age with low signal to noise ratio. The goal of this study is to use epigenetic age acceleration to develop early cancer risk detection, improve treatment options, and to understand the relationship between accelerated liver tissue aging and tumorigenesis. Epigenetic aging events are potentially reversible adjuvant. Methylated DNA is also far more stable in serum and tissue than un‐methylated nucleic acids. These factors make epigenetic events attractive candidates for serum and biopsy biomarkers.

Methods

Genomic DNA was extracted from IRB‐ approved, post‐operative or snap frozen, clinical patient liver resections or explants. DNA methylation (DNAm) was collected using Illumina BeadChip arrays. DNAm was used to calculate epigenetic age according to PCPhenoAge in over 600 liver samples, 8 tumor infiltrated NK‐cell isolates and 50 cell culture samples. PCPhenoAge is an improved version of the original biological aging clock with increased reliability and reduced susceptibility to technical noise. Multiple regression was used to residualize PCPhenoAge with respect to age and multiple phenotypes, such as alcohol consumption, known to affect epigenetic age. These residuals capture the epigenetic age acceleration independent of known behavioral risk factors for HCC.

Results

Preliminary analysis of 27 of our matched HCC patient tissues‐‐14 Alcohol and 13 Hepatitis C virus (HCV) associated HCC patients‐‐show higher PCPhenoAge in tumor versus non‐tumor tissues. Further, alcohol‐related HCC tumor tissue has significantly higher PCPhenoAge than HCV‐related HCC tumor tissue.
Pharmacokinetics of a 80mg Sublingual Dose of Delta‐Tocotrienol Powder
Authors: Wallace William A. , Gillespie Jonathan C. , Hodges Falyn L. , McCoun Julie A. , Raub Betsy J. , Serrano Eric R. , Moon Jordan R.
Published: May 2022

Collectively, tocopherols and tocotrienols are known as tocochromanols, a lipid‐soluble group of chemicals that make up the vitamin E family of compounds. There are four primary tocopherols (α‐tocopherol, β‐tocopherol, γ‐tocopherol, and δ‐tocopherol) and four primary tocotrienols (α‐tocotrienol, β‐tocotrienol, γ‐tocotrienol, δ‐tocotrienol). To date, tocotrienols have received less attention for their effects on mammalian health compared to their counterparts, tocopherols. Yet, δ‐tocotrienols (DT3) have shown efficacy in pre‐clinical anti‐cancer research. The purpose of this study was to investigate the pharmacokinetics and bioavailability of a powdered, sublingual form of DT3, in healthy men and women. We hypothesized that there would a steady increase in plasma DT3 followed by a decrease after reaching peak concentrations over an 8‐hour period. Volunteers (m=4, f=1, 30 +/‐ 4 years, 175 +/‐ 4 cm, 83.4 +/‐ 7.4 kg) were administered 80mg of DT3 powder in a fasted state. Volunteers were asked to keep the powder under their tongues until it was fully dissolved in two separate doses of 40mg given consecutively. Blood samples were taken at 0, 60, 90, 120, 180, 240, 300, 360, 420, and 480 minutes. Volunteers were given a meal consisting of carbohydrates, proteins, and fats after four hours. Blood samples collected were placed in a centrifuge to separate blood from plasma. Plasma was collected and stored in a ‐80° freezer. DT3 concentrations were calculated from plasma using protein precipitation followed by liquid‐liquid extraction. Analytes were separated by HPLC with the extracts assayed against a calibration curve. DT3 bioavailability was assessed using the parameters peak plasma concentration (Cmax), time to reach peak plasma concentration (Tmax) and total area under the plasma concentration‐time curve (AUC). Plasma concentration of DT3 reached 110.2 ng/ml (Cmax) 420 minutes after administration (Tmax). DT3 concentration decreased between 420 and 480 minutes. The 0‐8 h AUC reached 27,165.8 ng/ml*min. DT3 appears to exhibit a progressive, near linear upward absorption profile over the course of 7 hours, after which there is a decrease in plasma DT3.
Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 are Involved in Ritonavir‐induced Cytotoxicity in Hepatoma Cells
Authors: Wang Xiaofei , Yu Yihang , Wang Pei , Yang Kun , Yan Mengyao , Wang Yiting , Yan Liang , Zhong Xiao‐bo , Zhang Lirong
Published: May 2022

Ritonavir (RTV), a pharmacoenhancer used in anti‐HIV regimens, can induce liver damage. RTV is mainly metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A‐AS1) and HNF1A antisense RNA 1 (HNF1A‐AS1) are long noncoding RNAs (lncRNAs) that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and molecular mechanisms of HNF4A‐AS1 and HNF1A‐AS1 in RTV‐induced hepatotoxicity. Small hairpin RNAs were used to knock down HNF4A‐AS1 and HNF1A‐AS1 in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV‐induced hepatotoxicity. Chromatin immunoprecipitation qPCR was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A‐AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV‐induced cytotoxicity, whereas HNF1A‐AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A‐AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A‐AS1 knockdown. Alterations in RTV‐induced hepatotoxicity caused by decreasing HNF4A‐AS1 or HNF1A‐AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV‐induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A‐AS1 overexpression or HNF1A‐AS1 knockdown. Taken together, these results revealed that HNF4A‐AS1 and HNF1A‐AS1 modulated RTV‐induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter.