bluuebunny/crossref_metadata_2025 huggingface dataset:
Seizure Susceptibility Increases After Early‐Life Seizures in Zebrafish
Authors: Bhuiyan Kasulul , Ramon Alfonsina , Stegmuller Christopher , Lippman‐Bell Jocelyn
Published: May 2022

Early‐life seizures (ELS) can lead to the development of epilepsy in humans (Elger et al., 2004). Dynamic animal models are essential to study the effects of ELS in the developing brain. Zebrafish have emerged as a valuable model for neuroscience research due to their rapid development, the potential for high‐throughput screening, and their genetic similarity with mammals (Stewart et al., 2014). While rodent ELS studies show spontaneous seizures later in life (Stafstrom & Sasaki‐Adams, 2003), the effects of ELS on later‐life seizures in zebrafish has yet to be explored.

Based on human and rodent studies of ELS, we hypothesized that larval zebrafish would become more susceptible to seizures within two weeks after experiencing ELS. To test this, we induced ELS in larval zebrafish using the chemoconvulsant pentylenetetrazol (PTZ), a drug commonly used to induce seizures in zebrafish (Baraban et al., 2005). Zebrafish were exposed to 5mM PTZ for 40 minutes per day from 5‐7 days post‐fertilization (dpf). Clutchmate controls remained in a beaker inside an incubator for the duration of this time and were not exposed to PTZ. All fish were placed in a tank on an automated rack system at 8 dpf where they remained unperturbed until 21 dpf. At 21 dpf, zebrafish from both groups were pipetted into 12‐well plates where they were exposed to either 1, 2.5, or 5 mM PTZ for 20 minutes. For each condition and PTZ concentration, the number of stage two (whirlpool) seizures were measured.

In this pilot study, we found in 0%, 36.4%, and 50% of control fish demonstrated stage two seizures when administered 1, 2.5, and 5 mM PTZ at 21 dpf, respectively (n=8,11, and 12). However, 12.5%, 66.7%, and 66.7% of 21 dpf zebrafish that had previously experienced ELS had stage two seizures at those same concentrations, respectively (n = 8, 12, and 12). The number of stage two seizures demonstrated per fish were similar between conditions for 1mM and 5mM PTZ, but at 2.5 mM PTZ, zebrafish that experienced prior ELS had three times as many stage two seizures as controls (mean = 1.6 ± 0.5 SE for ELS fish, and 0.5 ± 0.2 SE for controls).

These observations provide evidence that larval zebrafish that experienced PTZ‐induced ELS as very young larvae (5‐7 dpf) may develop increased seizure susceptibility later in life. These results support the use of zebrafish in research aiming to identify the mechanisms by which ELS might lead to the development of epilepsy.
Cyclin C Suppresses Pancreatic Intraepithelial Neoplasia and Neural Endocrine Neoplasia Progression in a Murine Kras Model
Authors: Hanley Sara , Cai Kathy Q. , Stieg David C. , Santos Andre‐Klein , Campbell Kerry S. , Strich Randy
Published: May 2022

Cyclin C is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin C re‐localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin C suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin C in suppressing pancreatic cancer progression was investigated in the LSL‐KrasG12Dmurine cancer model. Within eight weeks, a 6‐8 fold increase in the appearance of pancreatic Intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions were observed in the KrasG12D+; Ccnc‐/‐ animals compared to KrasG12D alone. Surprisingly, high‐grade, poorly differentiated pancreatic neuroendocrine tumors/carcinomas (PNET/PNEC) were also observed. These lesions coexisted in the pancreas with some PanIN regions possessing intermixed cells expressing PNEC markers. Our previous studies identified a positive role for cyclin C in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+; Ccnc‐/‐ pancreas exhibited a reduction in autophagy compared to KrasG12D+; Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with elevated apoptosis. Consistent with this model, caspase 3 activation was observed in early KrasG12D+; Ccnc‐/‐PNETs but not in KrasG12D+ tissues. In addition, KrasG12D+; Ccnc‐/‐ cells showed decreased proteasomal activity and a 30% increase in cell death compared to KrasG12D+; Ccnc+/+ cells following treatment with the proteasome inhibitor, Bortezomib. Taken together, these results demonstrate a role for cyclin C in suppressing both PDAC and PNEN cancer progression and suggest a model that its loss promotes pre‐cancerous lesions by stimulating pancreatic cell injury.
Lipid droplets understood through student designed 3D printed models of the seipin protein complex
Authors: Juhler Isabelle G. , Mills Brooklyn D. , Kramer Lucas D. , Schumann Mia E. , Rodriguez‐Situ Maximilian J. , Hall Bonnie L.
Published: May 2022

Lipid droplets (LD’s) are organelles that play a key role in metabolism and human physiology, storing energy for cells to use when needed. Defects in LD metabolism can cause diseases such as obesity and lipodystrophy. LDs have a single phospholipid layer exterior and hold triglycerides and other lipids. Several different proteins are also found in the phospholipid exterior of an LD, for example GPAT (glycerol‐3‐phosphate O‐acyltransferase). LDs are assembled at the surface of the endoplasmic reticulum by the seipin protein complex, with the LD forming at the seipin site similar to “blowing a bubble”. We are undergraduate students at various levels in our training and have not encountered LDs in our coursework. However, we believe they are an important and intriguing model for teaching many introductory and more advanced biology and chemistry concepts. We designed a 3D printed model of the seipin complex (based on PDB file 6DS5, Yan 2018) that can be used in undergraduate teaching. This model, along with online videos and other tools, can be used to teach many topics through a hands‐on approach to learning. Topics we learned about while building our models and through the use of other materials included: protein structure, protein complexes, metabolism and its connection to different diseases, basic chemical structures, metabolic pathways, cellular structures, computational visualization of proteins, and the utilization of online tools such as the Protein Databank. LDs can be connected to content in many courses, including General Biology, General Chemistry, Biochemistry, Organic Chemistry, Genetics, Cell Biology and Physiology. Instructions for printing a copy of this model along with a list of other teaching resources can be made available to anyone who is interested.
Angico Gum suppresses inflammatory responses and maintains tissue integrity through the NO and COX‐2 pathway in intestinal mucositis induced by 5‐fluorouracil
Authors: Pimenta Helder , Barbosa Maria Lucianny , Medeiros Jand , Silva Durcilene , Vale Larice , Menslin Kimberlly , dos Santos Victor R. , Brendell John , Chaves Maryana , Russo Lisa M. , de Moraes Maria G. , Lopes Matheus , Brito Gerly A. , Cerqueira Gilberto
Published: May 2022

Intestinal homeostasis has often been referred to as a state of controlled inflammation, in which pro‐ and anti‐inflammatory factors function in a synchronous manner. In this context, intestinal mucositis can be deemed as an inflammatory pathophysiological process, which results in morphological and physiological changes in the intestinal mucosa of the gastrointestinal tract. Its emergence is due to the administration of antineoplastic agents, such as 5‐fluorouracil (5‐FU), an antimetabolite, during cancer therapy (1). With high rates of morbidity and mortality, mucositis still represents an obstacle in the treatment of cancer due to the lack of an effective treatment. Numerous studies have explored natural products and secondary metabolites from medicinal plants such as natural gums to find compounds having pharmacological activity against various human health disorders. Angico (Anadenanthera colubrina (Vell.)) is a plant of the Fabaceae family prevalent in the Brazilian ecosystems of the Cerrado and Caatinga (4). Angico has been reported to possess medicinal properties for the treatment of diarrhea, cough, bronchitis, and inflammation (5). Secondary metabolites such as steroids, flavonoids, terpenes, andphenolic derivatives can be extracted from the bark of A. colubrina (6) that can function as antioxidants, antimicrobials, and anti‐inflammatory, anti‐proliferative, antiallergic, antithrombotic, and antiviral agents (7). In the present study, we aimed to evaluate the effect of AG on experimental intestinal mucositis induced by 5‐FU. Swiss mice were randomly divided into nine groups: Saline, 5‐FU, AG‐100, AG‐400, Celecoxib (CLX), CLX + AG‐400, L‐NAME, L‐Arginine, and AG + L‐NAME. The animals were euthanized, then, blood and segments of the small intestine were collected to evaluate histopathological, levels of malondialdehyde (MDA) and Nitrite/Nitrate (NOx), leukocytes, mast and goblet cell counts, and immunohistochemical analysis of induced nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2). AG prevents morphofunctional changes in experimental intestinal mucositis due to 5‐FU by attenuating inflammation, oxidative stress, tissue damage, mastocytosis, and leukopenia. These results suggest that the NO and COX‐2 pathways are possible mechanisms mediating the protective effect of AG on intestinal mucositis induced by antineoplastic chemotherapeutic agents such as 5‐FU.
An upper limit to the fluorescence quantum yield for reduced flavins – a spectroscopic approach to better understand photoflavoproteins
Authors: McBride Rylee A. , Jacoby Morris Kimberly , Stanley Robert J.
Published: May 2022

Flavins are blue‐light absorbing chromophores with rich redox activity, found in oxidized, one electron‐ and two electron‐reduced forms. Some, namely flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are used by enzymes as catalytic cofactors. Conflicting literature regarding reduced flavin fluorescence emission includes a plethora of results, from some finding there is negligible fluorescence to others observing significantly blue‐shifted emission. This discrepancy is of consequence because mechanistic kinetic models of photoflavoproteins derived from optical spectroscopy rely on a proper assignment of this emission. Reduced flavins are common redox cofactors in cells, and any fluorescence‐based imaging of this molecule in vivo requires an accurate estimate of its emission quantum yield and spectrum. Here, the photo‐induced reduction of HPLC‐purified FMN and other relevant flavins, in aqueous solutions (at pH 5.0 and 8.5) under aerobic and anaerobic conditions, has been studied via steady‐state absorption and fluorescence measurements. An exogenous reducing agent, ethylenediaminetetraacetic acid (EDTA), was used to photo‐reduce the flavins under investigation. Facile and complete reduction of FMN (~105‐fold) is achieved under anaerobic conditions and insignificant amounts of reduced fluorescence emission is observed, allowing for calculation of the upper limit of the quantum yield of reduced flavin. The low quantum yield (~10‐4) obtained is an important finding when considering flavoproteins, especially when studying them spectroscopically.
Role of Human Aldehyde Oxidase in the Generation of Reactive Oxygen Species during the Metabolism of Nicotine
Authors: Kundu Tapan K. , Hemann Craig , Leimkühler Silke , Zweier Jay L.
Published: May 2022

Nicotine is the major addictive constituent of tobacco and alternative tobacco products including electronic cigarettes (e‐cigs). While e‐cig vaping has been proposed as safer than traditional tobacco smoking, recent studies has demonstrated that these products can also trigger oxidant stress and inflammation with resultant pulmonary and cardiovascular disease. E‐cig liquids contain high levels of nicotine typically of 12 to 24 mg/ml or more and with the use of high power delivery devices high, poorly controlled levels of nicotine can be delivered. When inhaling tobacco cigarette smoke or e‐cig vape, nicotine is rapidly absorbed through the lungs. The major pathway of nicotine metabolism in humans consists of the C‐oxidation of nicotine to produce cotinine involving a two‐step process: the first step is CYP2A6‐catalyzed 5′‐oxidation to nicotine‐Δ1′(5′)‐iminium ion (NI), which is in equilibrium with 5′‐hydroxynicotine; and the second step is aldehyde oxidase (AO) catalyzed oxidation of NI to cotinine.

Using both cytochrome‐c reduction and EPR spin trapping techniques, we show for the first time that the oxidation of NI by human AO produces superoxide radical anion (O2• −) with high flux rates. Purified recombinant human AO (hAO) was prepared and characterized by SDS gel electrophoresis and optical absorption spectroscopy. hAO, 40 nM was incubated in the presence of NI concentrations from 1‒50 µM. The kinetic parameters, Km and Vmax values, for superoxide generation from NI were determined to be 2.7 ± 0.4 μM and 694 ± 24 nmol min−1 mg−1 of protein, respectively. From quantitation of the measured superoxide and the amount of NI consumed, we estimate that ~60% of the oxygen consumed in this process is converted to superoxide with ~40% hydrogen peroxide. Superoxide generation was almost completely quenched by either 0.5 μM raloxifene, a potent inhibitor of AO, or 0.1 μM Copper‐Zinc‐superoxide dismutase (SOD1) with no superoxide generation detected either in the absence of either AO or nicotine‐Δ1′(5′)‐iminium ion.

Therefore, human AO efficiently catalyzes the oxidation of NI to cotinine with concomitant production of reactive oxygen species. Due to the high expression levels of AO in lung tissue as well as its rapid rate of NI metabolism with low Km, AO rapidly and efficiently converts NI to cotinine with the formation of superoxide and hydrogen peroxide. Thus, nicotine metabolism will be associated with the formation of superoxide, hydrogen peroxide and secondary reactive oxygen species that can in turn cause lung oxidative stress and inflammation.
Internalization of Angiotensin‐(1‐12) in Adult Retinal Pigment Epithelial‐19 Cells
Authors: Ahmad Sarfaraz , Wright Kendra N. , Ferrario Carlos M. , Cheng Heng‐Jie , Choudhary Mayur , Malek Goldis , Sappington Rebecca M.
Published: May 2022

Introduction

The characterization, by this laboratory, of angiotensin‐(1‐12) [Ang‐(1‐12)] as a primary angiotensin II (Ang II)‐forming substrate may be an unrecognized source of Ang II‐mediated microvascular complication in hypertension‐mediated retinopathy. We found that the plasma Ang‐(1‐12) level was 12‐66‐fold higher when compared to the angiotensin I in normal human subjects. For the first time, we investigated the endogenous expression and incorporation of Ang‐(1‐12) in cultured adult retinal pigment epithelial‐19 (ARPE‐19) cells. We also investigated the internalization of Ang‐(1‐12) in the presence of a highly specific monoclonal antibody (mAb) that was developed by us against the C‐terminus of the human Ang‐(1‐12) sequence.

Method

ARPE‐19 cells (passage 28‐30) were grown to post‐confluence in 10% serum supplemented DMEM/F12 medium for 2‐3 days. An immunofluorescence (IF) staining was performed to detect the internalized Ang‐(1‐12) using the human Ang‐(1‐12) mAb (1:5000 dilution) and the Alexa Flour 488 secondary antibody (1:1000 dilution). Internalization was also confirmed in ARPE‐19 using radiolabeled Ang‐(1‐12) [125I‐Ang‐(1‐12), purity >99%] in the presence and absence of the mAb by HPLC. For this, the APRE‐19 cells were incubated (4 h) with Ang‐(1‐12) alone or Ang‐(1‐12) + mAb. Afterward, cells were washed and harvested in isotonic buffer. The lysed cells were centrifuged at 28,000 g and the membrane pellet and supernatants (cytosolic fractions) were counted on a gamma counter. The cytosolic fractions were further analyzed by HPLC using a C18 column to confirm the internalization of intact Ang‐(1‐12) sequence.

Results

Our IF staining shows that Ang‐(1‐12) is endogenously expressed in ARPE‐19 cells. Further, an increased intensity of staining was detected in ARPE‐19 cells exposed to Ang‐(1‐12) (100 nM). High Ang‐(1‐12) radiolabeled counts were detected in the cytosolic fractions (3.1% of the total loaded radiolabeled) and very low counts in the membrane pellet (0.2%). We found that the internalization of 125I‐Ang‐(1‐12) was significantly decreased (~40%, P<0.0001) in the presence of the human Ang‐(1‐12) mAb (Fig. 1). The detection of a large peak (88%) of 125I‐Ang‐(1‐12) in the cytosolic fraction in the HPLC chromatogram further confirms that the intact Ang‐(1‐12) sequence is internalized by the cells (Fig. 2).

Conclusions

Ang‐(1‐12) is internalized by ARPE‐19 cells from extracellular spaces of the retina and the internalization of Ang‐(1‐12) decreased following exposure to the human Ang‐(1‐12) mAb. Overall our data suggests the human Ang‐(1‐12) mAb may be used as a novel therapeutic agent to prevent circulating Ang‐(1‐12) internalization into the retinal cells and subsequently reduce the intracellular generation of the Ang II from the Ang‐(1‐12) substrate.
BMI: A Screening Tool Analysis
Authors: Peltzer Cadynce , Khanna Depeesh , Kahar Payal , Parmar Mayur
Published: May 2022

Body mass index (BMI), a measurement based on a person’s height and weight, allows the classification of individuals into categories such as obese or overweight. With these classifications, we can assess risk for hypertension, diabetes, cancer, hypercholesterolemia, and other chronic diseases. Furthermore, childhood BMI serves as a prediction method for health and disease later in life. Along with BMI, researchers also study waist circumference and waist‐to‐hip ratio in correlation with the above‐mentioned chronic illnesses. This brief review explores the associations between body mass index, waist circumference, and the waist‐hip ratio as measurements and their capability as predictors for persistent conditions like diabetes and hypertension.
Biomimetic Collagen Membranes Functionalized with mRNA‐loaded Lipid Nanoparticles for Fibroblast Transdifferentiation to Endothelial Cells
Authors: Villarreal‐Leal Ramiro A. , Williams Michael E. , Taraballi Francesca , Cooke John P. , Corradetti Bruna
Published: May 2022

Non‐healing wounds are a common complication from chronic disorders such as diabetes, cardiovascular diseases and cancer, among others. Impaired vascularization during the early stages of the wound healing process is a major limitation to tissue repair, and contributes to the chronification of the injury, characterized by the deposition of fibrous tissue by fibroblasts. Targeted therapeutics aimed at enhancing vascularization at the site of injury by transdifferentiating fibroblasts to endothelial cells (EC) have the potential to promote healing and to prevent the wound from becoming chronic. Here we present a collagen membrane functionalized with phosphatidylserine (PS)‐containing lipid nanoparticles (LNP) that encapsulate mRNA encoding for proteins known to drive fibroblast transdifferentiation into EC (GATA2, ETV2, FLI1, KLF4). The nanoscopic particles we developed (~70 nm in size) show highly efficient RNA encapsulation rates (over 70%) and can be taken up by fibroblasts at rates similar to other conventional transfection methods such as lipofectamine (up to 40%). Gene expression of the delivered mRNA is also significantly increased in fibroblasts treated with our LNP. Collagen membranes were functionalized with Annexin V, a protein that binds the lipid PS, as an approach to attach our LNP onto the membranes and enhance their retention. Our results also show a successful functionalization of the collagen membranes with Annexin V and our LNP. These membranes could potentially be used as implants to promote in situ wound healing by acting as a niche whereby local fibroblasts are transdifferentiated to EC via uptake of the mRNA‐loaded LNP to enhance vascularization and expedite tissue repair. The biomimetic approach we propose here represents a novel alternative to the development of RNA therapeutics to concomitantly encapsulate and deliver ad hoc multiple RNAs, while protecting them from degradation and making it possible for them to exert specific biological functions in target cells. Furthermore, the versatility of these mRNA‐LNP formulations provides the foundation for targeting a variety of chronic diseases, with the potential of improving patient quality of life.
Contributions of the rostral vs. middle conchae in temporal countercurrent exchange in the domestic chicken
Authors: Poff Marlee , Owerkowicz Tomasz
Published: May 2022

The nasal conchae in extant endothermic vertebrates have been hypothesized to contribute to heat conservation during breathing at low ambient temperatures and heat dissipation at high ambient temperatures. In birds, the rostral and middle conchae in the nasal cavity increase surface area of the respiratory epithelium while decreasing the distance between the epithelium and the respiratory airstream. This improves the efficiency of heat and water transfer between them during temporal countercurrent exchange. To determine the role of each concha, we used surgical ablation to remove either the rostral conchae or both rostral and middle conchae in juveniles of the domestic chicken, as well as performing sham surgery on the control group (n=8, n=6, n=8, respectively). Using fast‐response thermocouples, we measured exhaled air temperature measurements at the external nares and glottis to determine heat and water savings in different portions of the upper respiratory tract. Measurements were taken in adult chickens under mild sedation within the ambient temperature (Ta) range of 10‐40°C and relative humidity of 30%. We expected that minimum exhaled air temperatures (Te) in animals which had undergone conchaectomies would be higher than those of the control group, especially in individuals with both the rostral and middle conchae removed. As expected, Ta had a significant effect (MANOVA, p<0.0001) on the efficiency of heat exchange in the nasal cavity, with the most pronounced (Ta‐Te) difference at the lowest Ta (10°C), and the least at the highest Ta (40°C). We also found surgical treatment had a significant effect, with double conchaectomised animals having higher Te’s than either the sham (p<0.05) or single conchaetomised (p<0.01) animals. Surprisingly, there was no significant difference between the latter two treatment groups. We found a significant interaction between Ta and surgical treatment (p<0.001), Ta and animal sex (p<0.01), and treatment and sex (p<0.05), Hens had slightly higher Te’s than roosters. Whether this was an effect of body mass, breathing rate, or cranial thermal windows (comb, wattle) remains to be determined. Overall, our data suggest that the middle conchae makes a significant contribution to the efficiency of the temporal countercurrent exchange in the nasal cavity, but the rostral conchae does not. Which begs the question – why did the rostral concha evolve?
Role of miRNA‐155 on tumorigenesis in a transgenic mouse model of breast cancer
Authors: McDonald Sierra J. , Cranford Taryn L. , VanderVeen Brandon N. , Cardaci Thomas , Daping Fan , Murphy Angela E.
Published: May 2022

Deregulated and aberrant expression of microRNAs (miRNAs) have been linked to a variety of disorders such as cancers, autoimmune diseases, and chronic inflammation. Many miRNAs are important regulators of cellular processes involving development, differentiation, and signaling. We investigated whether miR155, a multifunctional miRNA induced by inflammatory cytokines and involved in hematopoiesis, inflammatory disorders, and macrophage activation, has any promise in reducing tumor burden in a transgenic breast cancer model. To study the effects of miR155 in breast cancer, our lab developed the first MMTV‐PyMT mice deficient for miR155 on a C57BL/6 background. Four‐week‐old female PyMT (n=12) and PyMT/miR155‐/‐ (n=11) mice were euthanized at 19 weeks of age. miR155 deficient mice exhibited reduced tumor number and volume palpations (p<0.05) at approximately 16 weeks of age. Reduced tumor burden displayed in miR155 deficient mice was also confirmed by histopathological analysis. The majority (60%) of miR155 deficient mice exhibited a lower degree of dysplasia characterized as adenoma/mammary intraepithelial neoplasia (MIN), whereas all PyMT mice progressed into the early and late invasive carcinoma grade. Upon euthanizing, mammary glands were excised from tumors for RT‐PCR, and tumors were counted, measured, and weighed. PyMT mice deficient in miR155 exhibited reduced tumor volume (p<0.05), number (p<0.05), and weight (p<0.05). To investigate the anti‐tumoral effects associated with miR155 deficiency, mammary glands excised from tumors were analyzed to examine inflammatory mediators and macrophage markers within the mammary tumor environment. We demonstrate that miR155 deficiency upregulates suppressor of cytokine signaling 1 (SOCS1) resulting in reduced overall cytokines, and reduced macrophages as indicated by CD68 and Integrin Subunit Alpha X (IGTAX). Mammary glands of miR155‐/‐ mice had reduced pro‐inflammatory M1 macrophage cytokines as indicated by reduced interleukin‐1‐beta (IL‐1b), IL‐6, and tumor necrosis factor‐alpha (TNF‐a). Mammary glands excised from tumors of mice deficient in miR155 exhibited even greater inhibition of M2 macrophages, important in reducing inflammation and contributing to tumor growth and immunosuppressive function, as indicated by reduced IL‐4, IL‐10, IL‐13, mannose receptor 1 (Mrc1), and TNF‐beta. Given that macrophages are known to promote tumorigenesis, we suggest that the anti‐tumor effects of miR155 inhibition are due to its upregulation of SOCS1 and resulting decrease in pro‐tumor M2 macrophages.
Gross Anatomy of Bifid Xiphoid Process
Authors: Zamani Shahrzad , Meawad Sara , Van Der Horn Tamryn , Dhiman Shiv
Published: May 2022

Introduction

Xiphoid process is the ossified extension of the lower sternum in the chest midline of human adults. Natural variance in this process results in the less common bifurcated morphology.

Objective

The primary objective of this study was to explore the distribution of the bifid variant. As a secondary objective, we aimed to compare bifurcated Xiphoid processes to other possible variations.

Methods

A case study was designed to explore the distribution of bifid variants in a sample of cadavers (n=30) at a large medical education institution. The dependent variable was binarized (bifid or normal) and univariate analyses were performed based on the height, width, and presence of bifurcation measured in each cadaver.

Results

Data is reported using textual and diagrammatic visuals. A significant difference was observed between the measurements of the bifid and the normal variants. Additionally, we provide a review of the implications of this bony landmark for mediastinal pressure and surgeries performed on the thoracic and abdominal areas.

Conclusion

This case study demonstrates a significant variation of the Xiphoid process, aiding clinicians in performing more accurate imaging and diagnosis. Future research should consider the physiological effects and clinical significance of this process.
Role of Cytochrome P450s in 2,4,5‐Trichlorophenol Nephrotoxicity in Isolated Kidney Cells from Male Fischer 344 Rats
Authors: Rankin Gary O. , Torres Alex , Carter Carley , Vidrine David , Anestis Dianne K.
Published: May 2022

Trichlorophenols (TCPs) are used as chemical intermediates in the manufacture of a wide range of agricultural and industrial products. Previous studies have shown that 2,4,5‐TCP is the most potent nephrotoxicant in vitro of the six TCP isomers in isolated kidney cells (IKCs) from male Fischer 344 rats. It was also determined that 2,4,5‐TCP nephrotoxicity was attenuated by piperonyl butoxide, a non‐specific cytochrome P450 (CYP) inhibitor. The purpose of this study was to determine which CYPs might play a role in 2,4,5‐TCP nephrotoxicity in vitro in IKCs from male Fischer 344 rats. IKCs (~4 million/ml, 3 ml) were incubated with a selective CYP inhibitor or vehicle prior to addition of 2,4,5‐TCP (0.5 mM) or dimethyl sulfoxide (DMSO, 2,4,5‐TCP vehicle) for 60 min with shaking at 37oC under a 95% oxygen/5% carbon dioxide atmosphere. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the end of the incubation period. 2,4,5‐TCP‐induced cytotoxicity was completely attenuated by CYP inhibitors sulfaphenazole (CYP2C11) and thiotepa (CYP2B6). 2,4,5‐TCP cytotoxicity was also reduced by pretreatment with omeprazole (CYP2C19), DEDTCA (CYP2C/E), or isoniazid (CYP2E1). The least effective CYP inhibitor at reducing 2,4,5‐TCP cytotoxicity was oleandomycin (CYP3A). These results suggest that CYPs play a role in 2,4,5‐TCP nephrotoxicity in vitro with CYP2C11 and CYP2B6 being major contributors to promoting 2,4,5‐TCP cytotoxicity. The mechanism by which these and other CYPs promote 2,4,5‐TCP nephrotoxicity remains to be determined.
Divergent Anabolic Deficits and Pro‐inflammatory Effectors of Muscle Wasting in Xenograft and Metastatic Tumor Models of Colorectal Cancer
Authors: Kim Hyo‐Gun , Huot Joshua , Pin Fabrizio , Belcher Daniel , Bonetto Andrea , Nader Gustavo A.
Published: May 2022

Colorectal cancer (CRC) is the second deadliest cancer in the United States, with ~70% of CRC cases developing liver metastases and cachexia. Here we investigated two tumor types (HCT116 and C26) as xenograft (x) or metastatic (m) models. Male Nod SCID gamma and CD2F1 mice were injected subcutaneously or intrasplenically with HCT116 or C26 tumor cells, respectively. CRC tumor growth was investigated at 30 days (xHCT116), 24 days (mHCT116), and 14 days (xC26 and mC26), and resulted in a significant gastrocnemius muscle depletion regardless of tumor model or type (xHCT116: 14.2%, mHCT116: 29.0%, xC26: 20.4%, mC26: 17.1%; P < 0.05). mHCT116 tumors caused a reduction in ribosomal (r)RNA content (21.2%, P < 0.05) and rDNA transcription (40%, P < 0.001), but the mC26 tumor showed no loss of rRNA and the upregulation of rDNA transcription (>2‐fold, P < 0.001). All tumor‐bearing mice experienced a reduction in γ4E‐BP1 phosphorylation (xHCT116: 57.8%, mHCT116: 73.4%, xC26: 50.9%, mC26: 99.2%; P < 0.05) suggesting impaired translational control. Muscle IL‐6 mRNA was elevated in all tumor‐bearing mice except xHCT116 (mHCT116: 1.8‐fold, xC26: 3.6‐fold, mC26: 4.1‐fold; P < 0.05). TNF‐α mRNA was lower in xHCT116 (52.9%, P < 0.05) and was not induced in the other tumor‐bearing mice. Expression of the NOD‐, LRR‐ and pyrin domain‐containing protein 3 (NLRP3) was elevated only in mC26 muscle (5‐fold, P < 0.0001). Neither IL‐18 nor IL‐1β mRNA levels were consistent with changes in NLRP3, however IL‐1β mRNA increased in both tumor models and types (xHCT116: 5.5‐fold, mHCT116: 10‐fold, xC26: 2.5‐fold, mC26: 3.5‐fold; P < 0.05) with a greater expression in each metastatic relative to the xenograft model within each type of tumor. Our findings indicate that the mHCT116 tumor results in more drastic muscle depletion and anabolic deficits, while the mC26 exhibited a divergent pro‐inflammatory phenotype than the xenograft model. Our data highlight potentially important divergence in the pathogenesis of muscle wasting among the HCT116 and C26 pre‐clinical models of CRC.
Toll Like Receptor 4 Inhibition Improves Antitumor Responses to MBQ‐167 in Triple Negative Breast Cancer Cells in vitro
Authors: Grafals‐Ruiz Nilmary , Torres‐Sánchez Anamaris , Romero‐Vega Angélica C.
Published: May 2022

Triple negative breast cancer (TNBC) is an aggressive variant of breast cancer that lacks the expression of estrogen, progesterone, and human epithelial growth factor receptors. Due to the scarce availability of receptor‐targeted or hormonal treatments, TNBC patients are submitted to taxane‐based chemotherapies like paclitaxel. Patients initially respond to paclitaxel, but 4 out of 10 patients may develop tumor recurrence along with high metastasis and mortality rates. Several studies demonstrate that paclitaxel may induce chemoresistance and metastasis through the activation of Toll‐Like Receptor 4 (TLR4)/NFKB signaling pathway, a known cancer prognostic marker in breast cancer. Hence, new treatments are needed to improve TNBC prognosis in patients. Recently, our laboratory and collaborators developed MBQ‐167, a potent Rac and Cdc42 inhibitor that decreases tumor burden, cell proliferation, and metastasis in TNBC mouse models. In preliminary studies with TNBC mice, we observed that MBQ‐167/Paclitaxel combination treatment prevented metastasis progression compared to paclitaxel treatment alone. This led us to hypothesize that MBQ‐167 chemosensitizes aggressive breast cancer cells and reduces metastasis by blocking Rac/Cdc4, hence decreasing their downstream target NfkB activity. This research project aims to evaluate cellular and molecular events that explain how MBQ‐167 prevents paclitaxel‐induced metastasis. Here, we generated TLR4‐knockdowns (TLR4‐KD) in MDA‐MB‐231 TNBC cell lines using lentiviral particles with shRNAs against the TLR4 mRNA. After knockdown validation with western blot analysis (WB), Wildtype (MDA‐MB‐231), Scramble control knockdowns (Sc‐KD), and TLR4‐KD cells were treated with different concentrations of MBQ‐167, paclitaxel, doxorubicin, or combinations. After treatments, we evaluated the effect on cell viability from MTT assays at 96 and 120 hours, cell apoptosis through Caspase‐Glo 3/7 Assays at 48 hours, and cellular migration through scratch wound healing assays. Our results demonstrate that partial inhibition of TLR4 decreases the cell viability of cells, increases apoptosis, and decreases cell migration after treatment with MBQ‐167 (250 nM and 500 nM), Paclitaxel (5 nM and 10 nM), or Doxorubicin (250 nM and 500 nM). We also observed an additive response upon combinatorial treatments with MBQ‐167/Paclitaxel and MBQ‐167/Doxorubicin when TLR4‐KD cells were compared to wild‐type and Sc‐KD cells. Altogether, these results suggest that TLR4‐KD chemosensitizes cells to Paclitaxel and Doxorubicin chemotherapies. In addition, TLR4 inhibition improves cellular response to Rac/Cdc42 inhibitor, MBQ‐167. Therefore, TLR4‐KD may reduce Rac and NfkB signaling, improving treatment response to MBQ‐167, MBQ‐167/Paclitaxel, and MBQ‐167/Doxorubicin. Other cellular studies, including Rac and NfkB activation assays, are being evaluated in our laboratory.
Ovariectomy‐Induced Arterial Stiffening is Associated with Downregulation of Tissue Resident Macrophage Markers
Authors: Kilanowski‐Doroh Isabella , Ogola Benard , McNally Alexandra , D'Souza Shaina , Rojo Michelle , McLachlan James , Machado Heather , Lindsey Sarah
Published: May 2022

Tissue resident macrophages play a major role in the regulation of the extracellular matrix (ECM). The ECM provides an essential scaffold and mechanical stability for blood vessels, but remodeling due to acute injury or chronic inflammatory conditions like hypertension promotes vascular stiffening. We have previously shown that pulse wave velocity (PWV), a measure of arterial stiffness, is higher in male and ovariectomized (OVX) female mice versus intact female mice. RNA‐sequencing of aortas from intact and OVX mice revealed changes in several genes involved in fibrosis as well as macrophage function. Therefore, we hypothesized that estrogen protects from arterial stiffening through maintenance of “protective” macrophages and limiting pro‐fibrotic ECM components. Based on the RNAseq results, we assessed the impact of estrogen on aortic expression of four fibrosis‐associated genes: decorin (DCN), endothelin‐1 (Edn1), type III collagen α1 (Col3a1), and connective tissue growth factor (CTGF) as well as four cell surface macrophage markers: colony stimulating factor 1 receptor (CSF1R), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), AXL receptor tyrosine kinase (Axl), and growth arrest specific 6 (Gas6).

Methods

Female C57BL/6 mice were ovariectomized (OVX) at 8 weeks or remained intact. PWV was obtained as a measure of arterial stiffness using a Vevo 1100 high resolution ultrasound. Mice were euthanized between 20‐23 weeks of age for tissue collection. RNA was isolated from aortas using a Qiagen RNeasy Mini Kit and gene expression was quantified using droplet digital polymerase chain reaction (ddPCR). Statistical analysis was performed using an unpaired t‐test.

Results

Abdominal PWV was increased in OVX mice compared with intact mice (p<0.0001). Surprisingly, OVX induced a significant decrease (p=0.045) in DCN expression, while a downward trend was found for CTGF (p=0.10) and Col3a1 (p=0.08) but not Edn1 (p=0.59). OVX also decreased the expression of macrophage markers CSF1R (p=0.022) and LYVE1 (p=0.047), with a downward trend for Gas6 (p=0.096) but not Axl (0.161).

Conclusion

Increased PWV in OVX mice was not accompanied by upregulation of pro‐fibrotic genes but was associated with decreases in macrophage markers CSF1R and LYVE1 in the aorta. Therefore, estrogen loss may promote vascular stiffening through changes in the amounts or functions of specific subsets of macrophages in the vasculature. Our data support previous studies establishing a relationship between CSF1R, LYVE1, and arterial homeostasis. Future studies will examine the impact of estrogen on aortic tissue resident macrophages using flow cytometry and the downstream signaling that promotes arterial stiffening.
Comorbidity of Anemia and Otitis Media in Skeletal Remains
Authors: Gremba Allison
Published: May 2022

Within the clinical literature some researchers have identified a correlation between otitis media (OM) and anemia. OM is an inflammation or infection of the middle ear. Researchers have hypothesized that iron deficiency (anemia) reduces immune response and results in an increase in OM risk. For this study the comorbidity of OM and anemia was evaluated in the Hamann‐Todd Human Osteological Collection (n=20) and the Robert J. Terry Anatomical Skeletal Collection (n=105). The mastoid air cell system (MACS) hypopnuematization and porotic hyperostosis (PH) were used as skeletal markers of OM and anemia, respectively. A chi‐square test of independence between MACS hypopneumatization and PH was significant (p<0.013) (Table 1). These results lend some support to the limited clinical literature that reports anemia as a risk factor of OM, however, the relationship between anemia and OM is not discernable through skeletal remains. In addition, the results highlight the importance of comorbidities in skeletal analyses and anthropological researchers should aim to use a broad range of skeletal markers to answer questions about past populations.
Electronic Cigarette Exposure Induces Vascular Endothelial Dysfunction with NADPH Oxidase Activation and eNOS Uncoupling
Authors: Ewees Mohamed G. , El‐Mahdy Mohamed A. , Eid Mahmoud S. , Mahgoup Elsayed M. , Khaleel Sahar A. , Zweier Jay L.
Published: May 2022

Background

We recently reported a mouse model of chronic electronic cigarette (e‐cig) exposure‐induced cardiovascular pathology, where long‐term exposure to e‐cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Using this model, we performed studies to delineate the underlying mechanisms of ECV‐induced vascular endothelial dysfunction (VED), which is a central trigger of cardiovascular disease.

Methods

C57/BL6 male mice were exposed 2 hours/day, 5 days/week to ECV generated from e‐cig liquid containing 0, 6, or 24 mg/ml nicotine for 16 and 60 weeks. Blood pressure was measured by tail cuff and endothelial‐dependent and independent relaxation was measured on aortic segments using wire myography. Western blotting, immunofluorescence and ELISA were used to analyze the pathways involved in the mechanism of ECV‐induced VED.

Results

Time‐dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine‐induced aortic relaxation in ECV‐exposed mice, compared to air‐exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3‐nitrotyrosine levels in the aorta of ECV‐exposed mice were observed, indicating that ECV‐induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV‐induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt‐dependent eNOS phosphorylation occurred in the aorta of ECV‐exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV‐induced superoxide generation in the aorta of ECV exposed mice.

Conclusion

Our data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.
Modeling the role of cyclin C in connecting stress‐induced mitochondrial fission to apoptosis
Authors: Doyle Steven J. , Strich Randy
Published: May 2022

For normal cell function, exogenous signals must be correctly interpreted, and the proper response executed. The mitochondria are key regulatory nodes of cellular fate. For example, mitochondria undergo fission and fusion cycles depending on the energetic needs of the cell. Additionally, regulated cell death pathways also function at the mitochondria. Cyclin C is a transcriptional regulator of stress response and growth control genes. Following stress, a portion of cyclin C translocates to the cytoplasm, where it interacts with both the mitochondrial fission and apoptotic machinery. Based on these findings, we hypothesize that cyclin C represents a key mediator linking transcription to mitochondrial fission and intrinsic regulated cell death. Members of the cyclin protein family typically contain two conserved cyclin box domains each composed of five alpha‐helices. The N‐terminal cyclin box domain, CB1, is responsible for binding cyclin‐dependent kinases (Cdks), while the C‐terminal cyclin box, CB2, typically facilitates correct protein folding. For cyclin C, CB1 and CB2 mediate protein‐protein interactions with regards to transcriptional regulation (Cdk8) and mitochondrial fission (Drp1), respectively. Pull down assays show that pro‐apoptotic protein Bax interacts in a direct manner with cyclin C and this interaction is required for Bax activation and ultimately efficient intrinsic regulated cell death. Using the docking simulator ClusPro, cyclin C and Bax yielded consistent protein‐protein interaction sites within amino acids 160‐170 of cyclin C. Important residues in order of decreasing frequency include: Q161, D165, E169, Y162, Q164, and D170. Six sets of docking simulations yielded identical results, using multiple cyclin C and Bax protein data bank file combinations as a means of accounting for potential differences in the starting .pdb files. Inspection of this region in cyclin C shows a high degree of homology to the BH2 domain found in members of the Bcl‐2 protein family, such as Bax, Bak, and Bcl2. These data suggest that cyclin C possesses a BH2 domain directing Bax interaction and intrinsic regulated cell death. Interestingly, CB2 is also responsible for binding the fission protein Drp1. However preliminary modeling data suggest that this interaction is mediated by the latter half of CB2. Prior work also has demonstrated that while the BH2 domain is required for Bax binding, it is not required for interaction with Drp1. Co‐immunoprecipitation assays show Drp1 is required for cyclin C‐Bax interaction, suggesting a role for the interaction of all three proteins. Taken together, these results suggest a model that cyclin C physically bridges the fission and apoptotic machinery allowing the cell to coordinate mitochondrial dynamics with programmed cell death pathways.
Domestic crickets (Acheta domesticus) as an animal model to study Parkinson's disease
Authors: Arias‐Reyes Christian , Rojas‐Ruiz Andrés , Soliz Jorge
Published: May 2022

Impaired glutamatergic neurotransmission and neuronal metabolic dysfunction are classic alterations in the pathophysiology of Parkinson's disease (PD). The substantia nigra compacta of the brain (the area where the primary pathological lesion is located) is particularly exposed to oxidative stress and metabolic damage. A reduced ability to cope with metabolic demands, possibly related to impaired mitochondrial function, may make the substantia nigra highly vulnerable to the effects of glutamate, which acts as a neurotoxin in the presence of impaired cellular energy metabolism. Taking into account that insects and mammals share a similar molecular architecture in terms of brain function, this work investigated whether domestic crickets (Acheta domesticus) can be used as an animal model for the study of PD. For this, the crickets received an intra‐lymphatic injection (between the second and third tergit of the ventral abdominal segment) of glutamate (10 µl; 2M). Control animals received a similar volume of PBS solution. Twenty‐four hours after treatment, the brains of the animals were dissected. We use our oxygraph‐2K system (OROBOROS Instruments) to determine mitochondrial respiration by activating mitochondrial complexes (CI, CII, CI&II, and CIV) and the production of reactive oxygen species (ROS). Our preliminary results showed that glutamate treatment significantly reduced the respiration of mitochondrial complexes CI, CII, and CI&II, even though the activity of complex IV (an indicator of the number of mitochondria) was not reduced between treatments. On the other hand, mitochondrial production of reactive oxygen species (ROS) in glutamate‐treated animals was significantly increased when both mitochondrial complexes I&II were active, but this pattern was reversed when only one of the complexes I or II was active. Because similar alterations have been observed in the brains of mammals with Parkinson's disease, these results strongly suggest that domestic crickets can be used as an animal model to investigate the mitochondrial mechanisms involved in this disease.